肿瘤发生的肾虚端粒机制研究
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摘要
近年来,大量的临床研究结果证实,采用中医药辨证治疗肿瘤,能延长带瘤病人的生存期,提高病人的生活质量,改善临床症状,与放疗、化疗方案配合运用具有减毒增效的作用。以综合的临床疗效优势,我国肿瘤研究工作者提出了肿瘤疗效的综合评价体系,认为在肿瘤防治的临床实践中,应以人为本,容忍带瘤生存,注重延长病人的生存期,提高生活质量改善临床症状。这一新的医疗理念,是对以手术、放疗、化疗为主的以瘤体大小变化作为唯一标准衡量肿瘤疗效的评价体系的挑战,也使传统的以主观指标为主的中医临床疗效评价体系得以客观化。良好的临床疗效使众多的肿瘤患者从中受益,也引起了国内外肿瘤研究者的极大兴趣。因此,试图从天然药物中寻找抗肿瘤新药的研究成了一个新的热点。最先进的科学技术、最精密的实验仪器设备迅速被运用于中医药的研究中。筛选到了不少具有抗肿瘤功效的中草药,也从中草药中提取出了不少具有抗肿瘤作用的活性成分,中医药抗肿瘤的机制也得到了一一证实。然而,采用现代科技手段研究抗肿瘤中医药的结果,除了从另一个侧面更进一步证实中医药具有多层面、多环节、多靶点的全方位抗癌抑癌的综合疗效外,既未见到其对中、西医理论的促进作用,也未见到其对中、西医临床的指导作用,亦未见到其有形成新的医学理论的迹象。究其原因：1,中医临床注重的是因地因时因人制宜的个性化动态治疗方案,而西医实行的主要是群体化静态治疗方案；2,西医追求的是药物单体的药理功效,而中药对肿瘤机体起作用的是复方配伍和复合成分；3,西医临床以病为重,注重杀灭癌细胞,而中医以人为本,注重保持机体元气,容忍长期带瘤生存。由此可见,离开了中医理论指导下的任何研究,无论多先进、多科学,其结果必将陷入与临床脱离,与原有理论偏移,最终使学科退化的困境。要研究中医药抗肿瘤的机制,必须首先明确肿瘤发生的中医机制。
目的
1.用科学的实验数据把肿瘤发生的肾虚机制内涵表达出来。
2.建立具有科学属性的中医抗肿瘤理论,科学有效地指导抗肿瘤的临床实践和实验研究。
3.从细胞生物学和分子生物学水平丰富和发展中医理论。
方法与结果
1.通过观察右归饮含药血清对A549人肺腺癌细胞形态与功能、端粒动力学、端粒酶、端锚酶、P53基因的影响探讨肿瘤发生的肾虚端粒机制。
2.运用倒置显微镜观察A549人肺腺癌细胞形态、传代的变化,采用[3H]-TdR摄取试验和X-Gal转染试验检测衰老细胞,结果显示：经不同浓度右归饮含药血清处理后各组细胞对[3H]-TdR的摄取率逐渐降低,经7次传代后,30%右归饮含药血清组的摄取率降至：(6.78±0.92)%,10%、20%右归饮含药血清组分别降为：(37.49±2.68)％.(34.33±2.59)%,而对照组的摄取率为：(84.84±4.86)%,各组比较,差异显著(P<0.01)；各组细胞转染X-Gal的效率逐渐提高,经7次传代后,30%右归饮含药血清组X-Gal的转染率达到：(87.04±4.47)%,10%、20%右归饮含药血清组分别为：(34.55±2.75)％.(37.85±1.96)%,而对照组为：(6.20±0.89)%,各组比较,差异显著(P<0.01)；各组细胞每次传代时间逐渐延长,传代次数逐渐减少,衰老细胞逐渐显现,30%右归饮含药血清组在第7.00±0.63代,10%、20%组分别在第17.00±0.63代和15.50±0.84代,细胞传代终止并在显微镜下见衰老凋亡细胞。
3.采用定量荧光原位杂交法(Q-FISH)上流式细胞仪检测每一代细胞端粒长度,结果显示：经不同浓度右归饮含药血清处理后各组细胞端粒长度逐渐缩短,细胞传代丢失的端粒长度逐渐增加,30%右归饮含药血清组平均每次传代丢失的端粒长度达1.29±0.12kb,10%、20%组分别为0.53±0.11 kb和0.57±0.03kb。
4.运用多聚合酶链-酶联免疫吸附反应(PCR-ELISA)检测细胞端粒酶活性,结果显示：经不同浓度右归饮含药血清处理后各组端粒酶活性逐渐降低,在第7代培养细胞中,30%右归饮含药血清组端粒酶活性为36.44±2.49,10%、20%组分别为64.75±2.63和62.50±2.64,而对照组为87.50±3.05。
5.运用Western Blot反应检测端锚酶活性,结果显示：经不同浓度右归饮含药血清处理后各组端锚酶活性逐渐降低,在第7代培养细胞中,30%右归饮含药血清组在标准蛋白质分子量142ku区域端锚酶条带明显比其它两组变淡。
6.运用S-P免疫组化检测各组细胞P53蛋白(mtP53),结果显示：在400倍光镜下观察阳性A549细胞的数量和颜色,光线透过玻片上圆球形的细胞,细胞质、细胞器、细胞核清晰可见,突变型P53(mt P53)聚集于胞核周围,随着mt P53蛋白表达量的多少,核及核周围呈现棕褐色、棕黄色和黄色。对照组mt P53的表达最强,不同浓度的右归饮含药血清处理后,mt P53表达逐渐下降,以30%右归饮含药血清组下降最明显。根据观察到的阳性细胞数和颜色,综合评分结果显示：在对照组内,随机观察5个高倍视野,阳性细胞数大于75%,得4分,棕褐色,评3分,根据评分标准,综合评分在9到12分范围,mt P53的表达呈强阳性(+++)。
在10%和20%右归饮含药血清组内,随机观察5个高倍视野,阳性细胞数在51%到75%之间,得3分,棕黄色,评2分,根据评分标准,综合评分在5到8分范围,mt P53的表达呈中度阳性(++)。
在30%右归饮含药血清组内,随机观察5个高倍视野,阳性细胞数在26%到50%之间,评2分,棕黄色或黄色,得1到2分,根据评分标准,综合评分在1到4分范围,mt P53的表达呈弱阳性(+)。在第7代培养细胞中,30%右归饮含药血清组mtP53综合评分为3.78±0.94,10%、20%组分别为6.39±1.29和6.22±1.11,而对照组为11.50±1.15。
结论
1.肾主封藏的科学含义是：(1)保持机体能量的稳定,(2)保持细胞端粒时间、空间的稳定。
2.肾虚,封藏失职,机体能量失守是肿瘤发生的根本原因。
3.P53基因突变,端粒酶系统被激活,端粒的时、空不稳是肿瘤发生的端粒机制。
4.补肾可以增加肿瘤细胞每次传代丢失的端粒长度,保持端粒空间的稳定；减少肿瘤细胞传代次数,保持端粒时间的稳定。
5.补肾保持了端粒时、空的稳定,从而保持细胞、机体能量的稳定。
6.补肾可以抑制P53基因突变,抑制端粒酶活性,抑制永生化癌变细胞的发生。
7.肿瘤发生的肾虚端粒机制是肾虚,封藏失职,机体能量失守,端粒的时、空不稳,P53基因突变,端粒酶系统被激活,细胞获得无限增殖能力。
BACKGROUND
Recently, the treatment of tumor differently by traditional Chinese medicine(TCM),that can lengthen the life-span, improve the life quality,relax the clinic symptoms,weaken toxin and enhance effect with the program of radiotherapy and chemotherapy for the patients with tumor,has been confirmed by plenty of clinical research results.For the synthetic advantage of clinical effect, a synthetic evaluating system on tumor effect,that takes patients as the foremost,allows living with tumor,focuses on lengthening patient's life-span, improves life quality, alleviates clinical symptom in practice of preventing and treating tumor,has been proposed by tumor researchers.This new medical suggestion, that makes TCM evaluating system of clinical effect with main subjective index objectify, is a challenge to the evaluating system of measuring tumor curative effect with only standard of the change of tumor body treated mainly by operation, radiotherapy and chemotherapy. The excellent clinical effect benefits mass patients with tumor and arouses interest of tumor researchers at home and abroad. Therefore,the study of trying to search for new anti-tumor medicine from natural herbs has become a new popularity. The latest scientific technology, the most precision experimental instruments are promptly applied to the study of TCM. No less herbs with anti-tumor function have been screened out,many active elements with anti-tumor function have been extracted from herbs,the anti-tumor mechanism of TCM have been identified one by one.However, the results of studying anti-tumor Chinese medicine by modern means of science and technology can neither promote both Chinese and west medical theory, nor direct both clinical practices,nor create a new medical theory besides proving that Chinese medicine has synthetic effect on anti-carcinoma and restrain-carcinoma from multistage, multilink, multipoint.The reasons include:1.TCM focuses on individual dealing dynamic program of giving treatment in accordance with local conditions,seasonal conditions and patient's individuality, but west medicine mainly lays stress on static common treating program.2. West medicine seeks for pharmacological function of active facts,but it is compound and mixed elements in TCM that have effect on tumor body.3. West medicine takes illness seriously, aims at killing carcinoma cells, but TCM takes patients as the foremost,focuses on keeping the primordial QI in body, allows long-term existence with tumor.It proves that any study without directing of TCM theory, no matter how it is so advanced and scientific, will have to result in separating itself from clinical practice, deviating from original theory, eventually making subject degenerate. Undoubtedly,the TCM mechanism of tumor happening must be firstly defined before anti-tumor mechanism of TCM is studied.
OBJECTIVE This study is to determine the kidney-deficiency mechanism from telomere on tumor happening through observing the effect of serum containing YOUGUIYIN compound on morphology, telomere dynamics, the activities of telomerase and tankyrase,the expression of mtP53 in human lung adenocarcinoma A549 cells.
1.To express the kidney-deficiency mechanism of tumor happening by scientific data.
2.To build up the anti-tumor theory of TCM possessing scientific property, scientifically and effectively directs clinical practice and experimental study of anti-tumor.
3. To enrich and develop the TCM theory by the level of cell biology and molecular biology.
METHODS AND RESULTS
1.The kidney-deficiency mechanism from telomere on tumor happening is studied by observing the effect of YOUGUIYIN containing serum on morphology and function, telomere dynamics,the activities of telomerase and tankyrase and the expression of mtP53 in human lung adenocarnocima A549 cells.
2.The results that the changes of morphology and passage on human lung adenocarcinoma A549 cells are observed by inverted microscope, and the senescent cells are detected by the experiments about the uptake rate of human lung adenocarcinoma A549 cells to [3H]-TdR and the transfection efficiency of human lung adenocarcinoma A549 cells on X-Gal show:after dealing with different doses of YOUGUIYIN containing serum, the uptake rates of human lung adenocarcinoma A549 cells to[3H]-TdR become gradually decreased,after 7 passages,the uptake rates of human lung adenocarcinoma A549 cells to [3H]-TdR in 30% YOUGUIYIN containing serum group came down to(6.78±0.92)%,and to(37.49±2.68)%,(34.33±2.59)% respectively in 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group, but the uptake rates of human lung adenocarcinoma A549 cells to [3H]-TdR in control group was(84.84±4.86)%,the difference among groups was significant (P<0.01),the transfection efficiencies of human lung adenocarcinoma A549 cells on X-Gal become gradually increased in each group,after 7 passages,the transfection efficiencies on X-Gal in 30% YOUGUIYIN containing serum group came up to(87.04±4.47)%,and to (34.55±2.75) %,(37.85±1.96)% respectively in 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group,but the transfection efficiencies of human lung adenocarcinoma A549 cells on X-Gal in control group was(6.20±0.89)%,the difference among groups was significant (P<0.01),the duration of passage became gradually lengthened, the passage times became gradually reduced,and the senescence and apoptosis cells become gradually visible in each group, the passages end, the senescence and apoptosis cells appear in the 7th passage for 30% YOUGUIYIN containing serum group, and in the 17th and the 15th passage for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively,in the inverted microscope, appear the senescent and apoptotic cells,few of adherent cells growing, a large number of cell with gradually shrinking cell membrane due to dehydration, rare pseudopodia around the cells, the cell shape changing into flat and swell,that more particles can be seen within the cytoplasm, that the cell membrane was integrity, that the cell plasma became dense, that the dense organelles were visible, the more common cells with color fade chromatin, that the chromatin in nuclear change into dense, forming clumps with different shapes and sizes,coming to the boundary of the nuclear membrane,or the crescent bodies were formed, by Hoechst33342 staining,showing reduced nuclear due to dehydration, and irregular nuclei fragmentation phenomenon, nucleus cracking, multiple cytoplasm bud, that with the cytoplasmic bud off the sudden, the apoptotic body cells were formed.
3. The results that the telomere length in each passage cells are measured by Q-FISH show:after dealing with different doses of YOUGUIYIN containing serum, the telomere length becomes gradually shortened, the loses of telomere length in cell passing become gradually increased in each group,the loses of average telomere length in each passing for 30% YOUGUIYIN containing serum group reach 1.29±0.12kb, and 0.53±0.11kb,0.57±0.03kb for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively, compared with each group, showed statistically significant difference (P<0.01).
4.The results that the telomerase activities are measured by PCR-ELISA show:after dealing with different doses of YOUGUIYIN containing serum, the telomerase activity in each group becomes gradually reduced, the telomerase activity for 30% YOUGUIYIN containing serum group is 36.44±2.49,and 64.75±2.63, 62.50±2.64 for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively in the 7th passage cells,the difference among groups was significant (P<0.01).
5.The results that the tankyrase activities are measured by Western Blot show:after dealing with different doses of YOUGUIYIN containing serum, the tankyrase activity in each group becomes gradually reduced, the tankyrase label at the spot of 142ku molecular weight,standard protein, for 30% YOUGUIYIN containing serum group, fades markedly more than other two groups in the 7th passage cells.
6. The results that the mtP53 protein is measured by S-P immunohistochemistry show:the number and the colors of the positive A549 human lung adenocarcinoma cell with S-P immunohistochemical staining were observed in the ordinary optical microscope with magnification of 400 times,light through the spherical cell on the slide,the cytoplasm, organelle, the nucleus is clearly visible, mutant P53 gene protein(mtP53) mainly clustered around the nucleus,the colors of brown and red, brown and yellow, and yellow appear on the nucleus and around the nucleus in positive staining of A549 human lung adenocarcinoma cells with the number of the mtP53 protein expression, it can be seen that the expression of the mtP53 gene protein in the control group cell nucleus was the strongest,with different concentrations YOUGUIYIN containing serum treatment, the expression of mtP53 gene protein in A549 human lung adenocarcinoma cell nucleus gradually decreased,specially the expression of mtP53 gene protein in 30% YOUGUIYIN containing serum group was decreased most significantly. According to the observed number and colors of positive cells,the comprehensive evaluation results show that:in the control group,5 high power fields on each slide were observed, the number of positive cells was greater than 75%, score 4 points,the color intensity of staining is brown and red, score 3 points,according to rating standards,comprehensive evaluation was in the range of 9 to 12 points,indicating that mtP53 gene protein expression in the nucleus was strongly positive(+++).In 10% and 20% YOUGUIYIN containing serum group, five high power fields on each slide were observed, the number of positive cells was in 51% to 75%, and the score was 3 points,the color intensity of staining was brownish yellow, a score of 2 points, according to rating standards,integrated score was in the range of 5 to 8 points, indicating that mtP53 gene protein expression in the nucleus was moderately positive(++).In YOUGUIYIN 30% serum containing group, 5 high power fields on each slide were observed, the number of positive cells was in 26% to 50%, and a score of 2 points,stained brown and yellow or yellow color intensity, a score of 1 to 2 points,according to rating standards,integrated score range of 1 to 4, indicating that mtP53 gene protein expression in nucleus was weakly positive(+).After dealing with different doses of YOUGUIYIN containing serum, the expression of mtP53 protein in each group becomes gradually reduced, the synthetic score of mtP53 for 30% YOUGUIYIN containing serum group is 3.78±0.94,and 6.39±1.29,6.22±1.11 for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively in the 7th passage cells,but the score is 11.50±1.15,compared with each group, showed statistically significant difference (P<0.01).
CONCLUSIONS
1.The scientific implication for kidney dominating store-essence is: (1) keeping the energy of body steady, (2)keeping the space and the time of telomere in cell steady.
2.Kidney-deficiency, dysfunction of the kidney in storing essence, exhaustion of body energy, is the rooted reason of tumor happening.
3. Mutation of P53, being activated of telomerase system, unsteadying both space and time of telomere, is the telomere mechanism of tumor happening.
4.Reinforcing the kidney can increase the losses of telomere length of carcinoma cells in each passage so as to keep the space of telomere steady, and reduce carcinoma cell passages so as to keep the time of telomere steady.
5.Reinforcing the kidney can keep the space and the time of telomere steady so as to keep the energy of body and cells steady accordingly.
6. Reinforcing the kidney can inhibit P53 from mutating, repress telomerase activity, prevent immortal and carcinoma cells from happening.
7.Kidney-deficiency mechanism from telomere on tumor happening is that kidney-deficiency, dysfunction of the kidney in storing essence,exhaustion of body energy, unsteadying both space and time of telomere,mutation of P53,being activated of telomerase system, cell obtaining the ability to limitless proliferation.
目的
1.用科学的实验数据把肿瘤发生的肾虚机制内涵表达出来。
2.建立具有科学属性的中医抗肿瘤理论,科学有效地指导抗肿瘤的临床实践和实验研究。
3.从细胞生物学和分子生物学水平丰富和发展中医理论。
方法与结果
1.通过观察右归饮含药血清对A549人肺腺癌细胞形态与功能、端粒动力学、端粒酶、端锚酶、P53基因的影响探讨肿瘤发生的肾虚端粒机制。
2.运用倒置显微镜观察A549人肺腺癌细胞形态、传代的变化,采用[3H]-TdR摄取试验和X-Gal转染试验检测衰老细胞,结果显示：经不同浓度右归饮含药血清处理后各组细胞对[3H]-TdR的摄取率逐渐降低,经7次传代后,30%右归饮含药血清组的摄取率降至：(6.78±0.92)%,10%、20%右归饮含药血清组分别降为：(37.49±2.68)％.(34.33±2.59)%,而对照组的摄取率为：(84.84±4.86)%,各组比较,差异显著(P<0.01)；各组细胞转染X-Gal的效率逐渐提高,经7次传代后,30%右归饮含药血清组X-Gal的转染率达到：(87.04±4.47)%,10%、20%右归饮含药血清组分别为：(34.55±2.75)％.(37.85±1.96)%,而对照组为：(6.20±0.89)%,各组比较,差异显著(P<0.01)；各组细胞每次传代时间逐渐延长,传代次数逐渐减少,衰老细胞逐渐显现,30%右归饮含药血清组在第7.00±0.63代,10%、20%组分别在第17.00±0.63代和15.50±0.84代,细胞传代终止并在显微镜下见衰老凋亡细胞。
3.采用定量荧光原位杂交法(Q-FISH)上流式细胞仪检测每一代细胞端粒长度,结果显示：经不同浓度右归饮含药血清处理后各组细胞端粒长度逐渐缩短,细胞传代丢失的端粒长度逐渐增加,30%右归饮含药血清组平均每次传代丢失的端粒长度达1.29±0.12kb,10%、20%组分别为0.53±0.11 kb和0.57±0.03kb。
4.运用多聚合酶链-酶联免疫吸附反应(PCR-ELISA)检测细胞端粒酶活性,结果显示：经不同浓度右归饮含药血清处理后各组端粒酶活性逐渐降低,在第7代培养细胞中,30%右归饮含药血清组端粒酶活性为36.44±2.49,10%、20%组分别为64.75±2.63和62.50±2.64,而对照组为87.50±3.05。
5.运用Western Blot反应检测端锚酶活性,结果显示：经不同浓度右归饮含药血清处理后各组端锚酶活性逐渐降低,在第7代培养细胞中,30%右归饮含药血清组在标准蛋白质分子量142ku区域端锚酶条带明显比其它两组变淡。
6.运用S-P免疫组化检测各组细胞P53蛋白(mtP53),结果显示：在400倍光镜下观察阳性A549细胞的数量和颜色,光线透过玻片上圆球形的细胞,细胞质、细胞器、细胞核清晰可见,突变型P53(mt P53)聚集于胞核周围,随着mt P53蛋白表达量的多少,核及核周围呈现棕褐色、棕黄色和黄色。对照组mt P53的表达最强,不同浓度的右归饮含药血清处理后,mt P53表达逐渐下降,以30%右归饮含药血清组下降最明显。根据观察到的阳性细胞数和颜色,综合评分结果显示：在对照组内,随机观察5个高倍视野,阳性细胞数大于75%,得4分,棕褐色,评3分,根据评分标准,综合评分在9到12分范围,mt P53的表达呈强阳性(+++)。
在10%和20%右归饮含药血清组内,随机观察5个高倍视野,阳性细胞数在51%到75%之间,得3分,棕黄色,评2分,根据评分标准,综合评分在5到8分范围,mt P53的表达呈中度阳性(++)。
在30%右归饮含药血清组内,随机观察5个高倍视野,阳性细胞数在26%到50%之间,评2分,棕黄色或黄色,得1到2分,根据评分标准,综合评分在1到4分范围,mt P53的表达呈弱阳性(+)。在第7代培养细胞中,30%右归饮含药血清组mtP53综合评分为3.78±0.94,10%、20%组分别为6.39±1.29和6.22±1.11,而对照组为11.50±1.15。
结论
1.肾主封藏的科学含义是：(1)保持机体能量的稳定,(2)保持细胞端粒时间、空间的稳定。
2.肾虚,封藏失职,机体能量失守是肿瘤发生的根本原因。
3.P53基因突变,端粒酶系统被激活,端粒的时、空不稳是肿瘤发生的端粒机制。
4.补肾可以增加肿瘤细胞每次传代丢失的端粒长度,保持端粒空间的稳定；减少肿瘤细胞传代次数,保持端粒时间的稳定。
5.补肾保持了端粒时、空的稳定,从而保持细胞、机体能量的稳定。
6.补肾可以抑制P53基因突变,抑制端粒酶活性,抑制永生化癌变细胞的发生。
7.肿瘤发生的肾虚端粒机制是肾虚,封藏失职,机体能量失守,端粒的时、空不稳,P53基因突变,端粒酶系统被激活,细胞获得无限增殖能力。
BACKGROUND
Recently, the treatment of tumor differently by traditional Chinese medicine(TCM),that can lengthen the life-span, improve the life quality,relax the clinic symptoms,weaken toxin and enhance effect with the program of radiotherapy and chemotherapy for the patients with tumor,has been confirmed by plenty of clinical research results.For the synthetic advantage of clinical effect, a synthetic evaluating system on tumor effect,that takes patients as the foremost,allows living with tumor,focuses on lengthening patient's life-span, improves life quality, alleviates clinical symptom in practice of preventing and treating tumor,has been proposed by tumor researchers.This new medical suggestion, that makes TCM evaluating system of clinical effect with main subjective index objectify, is a challenge to the evaluating system of measuring tumor curative effect with only standard of the change of tumor body treated mainly by operation, radiotherapy and chemotherapy. The excellent clinical effect benefits mass patients with tumor and arouses interest of tumor researchers at home and abroad. Therefore,the study of trying to search for new anti-tumor medicine from natural herbs has become a new popularity. The latest scientific technology, the most precision experimental instruments are promptly applied to the study of TCM. No less herbs with anti-tumor function have been screened out,many active elements with anti-tumor function have been extracted from herbs,the anti-tumor mechanism of TCM have been identified one by one.However, the results of studying anti-tumor Chinese medicine by modern means of science and technology can neither promote both Chinese and west medical theory, nor direct both clinical practices,nor create a new medical theory besides proving that Chinese medicine has synthetic effect on anti-carcinoma and restrain-carcinoma from multistage, multilink, multipoint.The reasons include:1.TCM focuses on individual dealing dynamic program of giving treatment in accordance with local conditions,seasonal conditions and patient's individuality, but west medicine mainly lays stress on static common treating program.2. West medicine seeks for pharmacological function of active facts,but it is compound and mixed elements in TCM that have effect on tumor body.3. West medicine takes illness seriously, aims at killing carcinoma cells, but TCM takes patients as the foremost,focuses on keeping the primordial QI in body, allows long-term existence with tumor.It proves that any study without directing of TCM theory, no matter how it is so advanced and scientific, will have to result in separating itself from clinical practice, deviating from original theory, eventually making subject degenerate. Undoubtedly,the TCM mechanism of tumor happening must be firstly defined before anti-tumor mechanism of TCM is studied.
OBJECTIVE This study is to determine the kidney-deficiency mechanism from telomere on tumor happening through observing the effect of serum containing YOUGUIYIN compound on morphology, telomere dynamics, the activities of telomerase and tankyrase,the expression of mtP53 in human lung adenocarcinoma A549 cells.
1.To express the kidney-deficiency mechanism of tumor happening by scientific data.
2.To build up the anti-tumor theory of TCM possessing scientific property, scientifically and effectively directs clinical practice and experimental study of anti-tumor.
3. To enrich and develop the TCM theory by the level of cell biology and molecular biology.
METHODS AND RESULTS
1.The kidney-deficiency mechanism from telomere on tumor happening is studied by observing the effect of YOUGUIYIN containing serum on morphology and function, telomere dynamics,the activities of telomerase and tankyrase and the expression of mtP53 in human lung adenocarnocima A549 cells.
2.The results that the changes of morphology and passage on human lung adenocarcinoma A549 cells are observed by inverted microscope, and the senescent cells are detected by the experiments about the uptake rate of human lung adenocarcinoma A549 cells to [3H]-TdR and the transfection efficiency of human lung adenocarcinoma A549 cells on X-Gal show:after dealing with different doses of YOUGUIYIN containing serum, the uptake rates of human lung adenocarcinoma A549 cells to[3H]-TdR become gradually decreased,after 7 passages,the uptake rates of human lung adenocarcinoma A549 cells to [3H]-TdR in 30% YOUGUIYIN containing serum group came down to(6.78±0.92)%,and to(37.49±2.68)%,(34.33±2.59)% respectively in 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group, but the uptake rates of human lung adenocarcinoma A549 cells to [3H]-TdR in control group was(84.84±4.86)%,the difference among groups was significant (P<0.01),the transfection efficiencies of human lung adenocarcinoma A549 cells on X-Gal become gradually increased in each group,after 7 passages,the transfection efficiencies on X-Gal in 30% YOUGUIYIN containing serum group came up to(87.04±4.47)%,and to (34.55±2.75) %,(37.85±1.96)% respectively in 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group,but the transfection efficiencies of human lung adenocarcinoma A549 cells on X-Gal in control group was(6.20±0.89)%,the difference among groups was significant (P<0.01),the duration of passage became gradually lengthened, the passage times became gradually reduced,and the senescence and apoptosis cells become gradually visible in each group, the passages end, the senescence and apoptosis cells appear in the 7th passage for 30% YOUGUIYIN containing serum group, and in the 17th and the 15th passage for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively,in the inverted microscope, appear the senescent and apoptotic cells,few of adherent cells growing, a large number of cell with gradually shrinking cell membrane due to dehydration, rare pseudopodia around the cells, the cell shape changing into flat and swell,that more particles can be seen within the cytoplasm, that the cell membrane was integrity, that the cell plasma became dense, that the dense organelles were visible, the more common cells with color fade chromatin, that the chromatin in nuclear change into dense, forming clumps with different shapes and sizes,coming to the boundary of the nuclear membrane,or the crescent bodies were formed, by Hoechst33342 staining,showing reduced nuclear due to dehydration, and irregular nuclei fragmentation phenomenon, nucleus cracking, multiple cytoplasm bud, that with the cytoplasmic bud off the sudden, the apoptotic body cells were formed.
3. The results that the telomere length in each passage cells are measured by Q-FISH show:after dealing with different doses of YOUGUIYIN containing serum, the telomere length becomes gradually shortened, the loses of telomere length in cell passing become gradually increased in each group,the loses of average telomere length in each passing for 30% YOUGUIYIN containing serum group reach 1.29±0.12kb, and 0.53±0.11kb,0.57±0.03kb for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively, compared with each group, showed statistically significant difference (P<0.01).
4.The results that the telomerase activities are measured by PCR-ELISA show:after dealing with different doses of YOUGUIYIN containing serum, the telomerase activity in each group becomes gradually reduced, the telomerase activity for 30% YOUGUIYIN containing serum group is 36.44±2.49,and 64.75±2.63, 62.50±2.64 for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively in the 7th passage cells,the difference among groups was significant (P<0.01).
5.The results that the tankyrase activities are measured by Western Blot show:after dealing with different doses of YOUGUIYIN containing serum, the tankyrase activity in each group becomes gradually reduced, the tankyrase label at the spot of 142ku molecular weight,standard protein, for 30% YOUGUIYIN containing serum group, fades markedly more than other two groups in the 7th passage cells.
6. The results that the mtP53 protein is measured by S-P immunohistochemistry show:the number and the colors of the positive A549 human lung adenocarcinoma cell with S-P immunohistochemical staining were observed in the ordinary optical microscope with magnification of 400 times,light through the spherical cell on the slide,the cytoplasm, organelle, the nucleus is clearly visible, mutant P53 gene protein(mtP53) mainly clustered around the nucleus,the colors of brown and red, brown and yellow, and yellow appear on the nucleus and around the nucleus in positive staining of A549 human lung adenocarcinoma cells with the number of the mtP53 protein expression, it can be seen that the expression of the mtP53 gene protein in the control group cell nucleus was the strongest,with different concentrations YOUGUIYIN containing serum treatment, the expression of mtP53 gene protein in A549 human lung adenocarcinoma cell nucleus gradually decreased,specially the expression of mtP53 gene protein in 30% YOUGUIYIN containing serum group was decreased most significantly. According to the observed number and colors of positive cells,the comprehensive evaluation results show that:in the control group,5 high power fields on each slide were observed, the number of positive cells was greater than 75%, score 4 points,the color intensity of staining is brown and red, score 3 points,according to rating standards,comprehensive evaluation was in the range of 9 to 12 points,indicating that mtP53 gene protein expression in the nucleus was strongly positive(+++).In 10% and 20% YOUGUIYIN containing serum group, five high power fields on each slide were observed, the number of positive cells was in 51% to 75%, and the score was 3 points,the color intensity of staining was brownish yellow, a score of 2 points, according to rating standards,integrated score was in the range of 5 to 8 points, indicating that mtP53 gene protein expression in the nucleus was moderately positive(++).In YOUGUIYIN 30% serum containing group, 5 high power fields on each slide were observed, the number of positive cells was in 26% to 50%, and a score of 2 points,stained brown and yellow or yellow color intensity, a score of 1 to 2 points,according to rating standards,integrated score range of 1 to 4, indicating that mtP53 gene protein expression in nucleus was weakly positive(+).After dealing with different doses of YOUGUIYIN containing serum, the expression of mtP53 protein in each group becomes gradually reduced, the synthetic score of mtP53 for 30% YOUGUIYIN containing serum group is 3.78±0.94,and 6.39±1.29,6.22±1.11 for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively in the 7th passage cells,but the score is 11.50±1.15,compared with each group, showed statistically significant difference (P<0.01).
CONCLUSIONS
1.The scientific implication for kidney dominating store-essence is: (1) keeping the energy of body steady, (2)keeping the space and the time of telomere in cell steady.
2.Kidney-deficiency, dysfunction of the kidney in storing essence, exhaustion of body energy, is the rooted reason of tumor happening.
3. Mutation of P53, being activated of telomerase system, unsteadying both space and time of telomere, is the telomere mechanism of tumor happening.
4.Reinforcing the kidney can increase the losses of telomere length of carcinoma cells in each passage so as to keep the space of telomere steady, and reduce carcinoma cell passages so as to keep the time of telomere steady.
5.Reinforcing the kidney can keep the space and the time of telomere steady so as to keep the energy of body and cells steady accordingly.
6. Reinforcing the kidney can inhibit P53 from mutating, repress telomerase activity, prevent immortal and carcinoma cells from happening.
7.Kidney-deficiency mechanism from telomere on tumor happening is that kidney-deficiency, dysfunction of the kidney in storing essence,exhaustion of body energy, unsteadying both space and time of telomere,mutation of P53,being activated of telomerase system, cell obtaining the ability to limitless proliferation.
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