大蒜素对宫颈癌Hela细胞HPV18-E6 p53 hTERT基因表达影响的研究
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摘要
目的: 宫颈癌是危害妇女健康的常见恶性肿瘤，死亡率为妇科恶性肿瘤之首，从宫颈癌前病变发展成为宫颈癌是一个较为漫长的过程，大约需要十年甚至更长，因此，宫颈癌是一个可预防、可治愈的疾病。及时发现早期宫颈癌，及时恰当的处理，治愈率几乎达100%。因此，在宫颈病变的早期及时阻断其向恶性方向发展，防止宫颈癌发生，具有重要意义。现已证实人乳头瘤病毒（Human papilloma virus，HPV）是宫颈癌的重要致病因素，但由于HPV病毒尚未能在体外成功培养，故关于宫颈癌的预防性研究相对滞后。本实验在前人研究的基础上，筛选一种诱导宫颈癌细胞凋亡的药物——大蒜素，观察其是否能对整合入Hela细胞中的HPV18病毒基因序列中的癌基因E6、 P53和端粒酶的表达产生影响，以此为基础来推测该种药物能否对HPV病毒基因序列中的E6基因产生影响，从一个新的角度对HPV病毒的生物学性状进行研究。为宫颈癌的临床预防、治疗提供实验室理论依据。
方法
1细胞培养：常规方法复苏冻存于液氮罐中的人宫颈癌Hela细胞，接种于含10%胎牛血清RPMI~1640培养液的培养瓶中，置于37℃、5%二氧化碳饱和湿度培养箱内培养，每天观察细胞生长情况，待细胞生长至70%融合时（约2~3
天），EDTA消化，制成单细胞悬液，以备实验用。
2 MTT比色法测定细胞生长抑制情况：调整细胞密度至3×104个细胞/ ml，充分混匀，接种到96孔培养板中，每孔200μl，置于培养箱内培养，至对数生长期，吸去上清液，实验组分别加入200μl新鲜配制的不同浓度的大蒜素培养液，浓度分别为25mg/L、 50 mg/L、 100 mg/L，对照组加入200μl培养液。每天于显微镜下观察细胞生长情况，分别于24小时、48小时、72小时取出培养板，每孔加入20μl（浓度为5g/L）MTT，轻振培养板，置培养箱中孵育4小时后，弃去上清液，加入二甲基亚砜200μl，室温震荡8分钟，使其彻底溶解，于酶标仪570nm波长处测其吸光度A值，计算出抑制率（IR）。同法重复实验3次。抑制率（IR）=（1–实验组A值/对照组A值）×100%。
3光学显微镜观察细胞形态：调整细胞密度至3×104个细胞/ ml，接种到预置盖玻片的24孔培养板中，每孔1ml。置于培养箱内培养，至对数生长期，更换培养液，加入药物。实验组加入终浓度分别为25mg/L、 50 mg/L、 100 mg/L、200 mg/L的大蒜素，同样设对照组，12小时、18小时、24小时、48小时、72小时后取出盖玻片，Gimsa染色，光镜下观察细胞形态。
4透射电镜观察细胞形态：将细胞按照上述浓度接种于培养瓶中，上述浓度药物作用12小时、18小时、24小时、48小时、72小时后，收集细胞，2.5%戊二醛前固定，清洗后1%锇酸后固定，常规脱水，浸透，环氧树脂包埋，切成超薄切片，透射电子显微镜观察细胞形态。
5 TUNEL法检测细胞凋亡：按照光学显微镜观察细胞
形态方法获得细胞爬片，置于培养箱内培养，至对数生长期，更换培养液，加入药物。药物浓度同上，设对照组。24小时、48小时、72小时后，按照TUNEL试剂盒说明操作，对细胞爬片进行处理，细胞核染成棕褐色者为凋亡细胞，核蓝色者为阴性。每个视野计数100个细胞，每张片计数3个视野，阳性细胞数占细胞总数的百分率即为凋亡指数（AI）。
6用PCR和RT–PCR方法研究HPV18 E6基因、P53基因、hTERT基因表达在细胞凋亡过程中的变化：用Trizol提取细胞总RNA。取扩增产物，经1.5％琼脂糖凝胶电泳（含EB），90V，45分钟，在紫外灯下观察结果，并在读胶仪AlphaImager1200上进行扫描，观察条带的灰度强弱，结果以目的基因与β–actin的积分吸光度的比值表示。
结果
1光学显微镜下细胞形态学改变：Hela细胞形状不规则，可为多角形、梭形等，核仁2~3个。大蒜素作用48小时，经Gimsa染色，光镜下可见细胞体缩小，胞膜完整，核染色质致密固缩，聚集于细胞核一侧，呈新月形；聚集于核的两端，呈八字形；聚集于整个核膜，呈周边化；或者可见到细胞核碎裂为花瓣状、大小不等的核片断。
2三种浓度大蒜素作用于细胞24小时、48小时、72小时细胞生长受到不同程度的抑制，明显高于对照组；各浓度间及各时间抑制率差异显著（P <0.05）。呈剂量、时间依赖性。72小时抑制率分别为38.46%、56.25%、65.32%、76.33%。
3透射电子显微镜下细胞形态学改变：实验组细胞呈现细胞凋亡样改变: 表现为细胞体积变小，细胞微绒毛消失，
胞内大量空泡形成；常染色质增加，核质比例减小；部分细胞呈典型的凋亡改变：核膜内陷，不规则，细胞染色质边集，固缩，紧靠核膜，聚居于细胞核核膜下，呈境界分明的团块状、花瓣状，细胞核碎裂、固缩，胞质呈片状脱落。随着药物浓度的增加及时间的延长愈加明显。对照组凋亡现象不明显，染色质分布均匀，可见1~2个核仁。
4 TUNEL标记结果：大蒜素作用不同时间后，凋亡细胞胞核经TUNEL染色呈棕褐色，核固缩、染色质凝集成块儿或边集。三种浓度大蒜素作用24、48、72小时细胞凋亡率均明显高于对照组（P <0.01），各浓度间凋亡率比较差异亦有显著意义（P <0.01）。
5大蒜素对Hela细胞株中HPV18E6、hTERT表达的影响：用RT-PCR检测Hela细胞株中HPV18E6基因表达，对照组和实验组Hela细胞均在352bp处扩增出?
Objective: Cervix lesion is one of the most common suffering in women, and it can develop to cervical carcinoma. In female carcinoma, the incidence of cervical carcinoma is the second only to mammary cancer. In our country, cervical carcinoma is very a common malignant tumor. It may take ten years of more from cervix lesion developing to cervical cancer. So we believe that it was a kind of cancer could be prevented and cured if diagnosed and properly treated early. It is very important to prevent it’s developing in early time. It is well known that HPV（human papilloma virus）is the initiation factor in the occurrence of the cervical cancer. Up to now, HPV cannot be cultivated in vitro, because of which, the research about preventing cervical cancer is falled behind. Based on previously study, we want to study the effect of allacin on gene expression of HPV18-E6, hTERT and P53 of Hela cell in vitro. The purpose of this study is to provide a new way to prevent and cure the cervical cancer in clinic.
Methods
1 Cell culture: Hela cells freezed in liguid nitrogen were
revived in routine method, and inoculated in RPMI-1640 blended with 10% foetus cattle serum, cultured in culture bottle. The bottle was put in the incubator with the condition of 37℃、5%CO2. The growth of cells was observed every day. 70% of the cells confluence was digested with EDTA and then was made into cell suspension.
2 The growth suppression of cell was estimated by MTT: adjusted deuterium of cells to 3×104 / ml, mixed them completely and inoculated them to cultivation Plate of 96–pore, 200μl in every pore. Cells were put to incubator and cultured to logarithm phrase. Then they were got out , the superior liquor was got rid of and 200μl culture medium including drug was added. The concentration of drug are 25mg/L、 50 mg/L、 100 mg/L. Then they were put back to incubator, watched everyday. 20μl MTT was added in every pore after cultured 24、48、72 hours, cultured in incubator again for 4 hours. Then, all liquor was got rid of. 200μl di–methyl–Sulfoxide was added, shocked for 8 minutes. And then the number of A and calculated the inhibition rate were measured. The sequences were done for 3 times in the same way.
3 Observation by inverted microscope. Hela cell suspensions were seeded in 24~well culture plates at a density of 3×104 cells/ml (1ml/well) in culture medium. Experimental treatment of cells was same as above. 24 and 72 h later, cells were observed and shot by inverted microscope. Observation after Giemsa staining by light microscope. Hela cell suspensions were seeded in 24-well culture plates at a density of 3 x 104 cells/ml (1ml/well) in culture
medium with a small piece of cover glass in each well. Experimental treatment of cells was same as above. 72 h later, the glass was taken out and stained with Giemsa stain for 10 to 15 min. Cells were observed by light microscope.
4 Observation by electron microscope. Hela cell suspensions were seeded in 100ml culture flasks. Experimental treatment of cells was same as above. 72 h later, the cells were harvested by treatment with EDTA(0.02%) and fixed by glutaral(4%) at 4 ℃. After desiccation, penetration, embedding, section and staining, cells were observed by electron microscope (JEM-1230, Japan).
5 Assayed by TUNEL Test：Experimental treatment of cells was same as above. The cells were treated according to instruction of TUNEL Test Kit. The cells whose nucleuses were stained by brown were apoptosis cells, and the blue are negative. 300 cells were counted in every section, and then calculated the apoptosis rate of cells.
6 With PCR and RT-PCR, the expression change of HPV18E6 mRNA, p53 mRNA as well as hTERT mRNA were studied in the process of Hela cell apoptosis in vitro.
Results
1. Observation after Giemsa staining by light microscope：After Giemsa staining, cytoplasm of Hela cells in untreated group was pink and nucleus the number of which was two or three were royal blue. Cell membrane and nucleus were integrated. More karyokinesis and few chromatin margi
方法
1细胞培养：常规方法复苏冻存于液氮罐中的人宫颈癌Hela细胞，接种于含10%胎牛血清RPMI~1640培养液的培养瓶中，置于37℃、5%二氧化碳饱和湿度培养箱内培养，每天观察细胞生长情况，待细胞生长至70%融合时（约2~3
天），EDTA消化，制成单细胞悬液，以备实验用。
2 MTT比色法测定细胞生长抑制情况：调整细胞密度至3×104个细胞/ ml，充分混匀，接种到96孔培养板中，每孔200μl，置于培养箱内培养，至对数生长期，吸去上清液，实验组分别加入200μl新鲜配制的不同浓度的大蒜素培养液，浓度分别为25mg/L、 50 mg/L、 100 mg/L，对照组加入200μl培养液。每天于显微镜下观察细胞生长情况，分别于24小时、48小时、72小时取出培养板，每孔加入20μl（浓度为5g/L）MTT，轻振培养板，置培养箱中孵育4小时后，弃去上清液，加入二甲基亚砜200μl，室温震荡8分钟，使其彻底溶解，于酶标仪570nm波长处测其吸光度A值，计算出抑制率（IR）。同法重复实验3次。抑制率（IR）=（1–实验组A值/对照组A值）×100%。
3光学显微镜观察细胞形态：调整细胞密度至3×104个细胞/ ml，接种到预置盖玻片的24孔培养板中，每孔1ml。置于培养箱内培养，至对数生长期，更换培养液，加入药物。实验组加入终浓度分别为25mg/L、 50 mg/L、 100 mg/L、200 mg/L的大蒜素，同样设对照组，12小时、18小时、24小时、48小时、72小时后取出盖玻片，Gimsa染色，光镜下观察细胞形态。
4透射电镜观察细胞形态：将细胞按照上述浓度接种于培养瓶中，上述浓度药物作用12小时、18小时、24小时、48小时、72小时后，收集细胞，2.5%戊二醛前固定，清洗后1%锇酸后固定，常规脱水，浸透，环氧树脂包埋，切成超薄切片，透射电子显微镜观察细胞形态。
5 TUNEL法检测细胞凋亡：按照光学显微镜观察细胞
形态方法获得细胞爬片，置于培养箱内培养，至对数生长期，更换培养液，加入药物。药物浓度同上，设对照组。24小时、48小时、72小时后，按照TUNEL试剂盒说明操作，对细胞爬片进行处理，细胞核染成棕褐色者为凋亡细胞，核蓝色者为阴性。每个视野计数100个细胞，每张片计数3个视野，阳性细胞数占细胞总数的百分率即为凋亡指数（AI）。
6用PCR和RT–PCR方法研究HPV18 E6基因、P53基因、hTERT基因表达在细胞凋亡过程中的变化：用Trizol提取细胞总RNA。取扩增产物，经1.5％琼脂糖凝胶电泳（含EB），90V，45分钟，在紫外灯下观察结果，并在读胶仪AlphaImager1200上进行扫描，观察条带的灰度强弱，结果以目的基因与β–actin的积分吸光度的比值表示。
结果
1光学显微镜下细胞形态学改变：Hela细胞形状不规则，可为多角形、梭形等，核仁2~3个。大蒜素作用48小时，经Gimsa染色，光镜下可见细胞体缩小，胞膜完整，核染色质致密固缩，聚集于细胞核一侧，呈新月形；聚集于核的两端，呈八字形；聚集于整个核膜，呈周边化；或者可见到细胞核碎裂为花瓣状、大小不等的核片断。
2三种浓度大蒜素作用于细胞24小时、48小时、72小时细胞生长受到不同程度的抑制，明显高于对照组；各浓度间及各时间抑制率差异显著（P <0.05）。呈剂量、时间依赖性。72小时抑制率分别为38.46%、56.25%、65.32%、76.33%。
3透射电子显微镜下细胞形态学改变：实验组细胞呈现细胞凋亡样改变: 表现为细胞体积变小，细胞微绒毛消失，
胞内大量空泡形成；常染色质增加，核质比例减小；部分细胞呈典型的凋亡改变：核膜内陷，不规则，细胞染色质边集，固缩，紧靠核膜，聚居于细胞核核膜下，呈境界分明的团块状、花瓣状，细胞核碎裂、固缩，胞质呈片状脱落。随着药物浓度的增加及时间的延长愈加明显。对照组凋亡现象不明显，染色质分布均匀，可见1~2个核仁。
4 TUNEL标记结果：大蒜素作用不同时间后，凋亡细胞胞核经TUNEL染色呈棕褐色，核固缩、染色质凝集成块儿或边集。三种浓度大蒜素作用24、48、72小时细胞凋亡率均明显高于对照组（P <0.01），各浓度间凋亡率比较差异亦有显著意义（P <0.01）。
5大蒜素对Hela细胞株中HPV18E6、hTERT表达的影响：用RT-PCR检测Hela细胞株中HPV18E6基因表达，对照组和实验组Hela细胞均在352bp处扩增出?
Objective: Cervix lesion is one of the most common suffering in women, and it can develop to cervical carcinoma. In female carcinoma, the incidence of cervical carcinoma is the second only to mammary cancer. In our country, cervical carcinoma is very a common malignant tumor. It may take ten years of more from cervix lesion developing to cervical cancer. So we believe that it was a kind of cancer could be prevented and cured if diagnosed and properly treated early. It is very important to prevent it’s developing in early time. It is well known that HPV（human papilloma virus）is the initiation factor in the occurrence of the cervical cancer. Up to now, HPV cannot be cultivated in vitro, because of which, the research about preventing cervical cancer is falled behind. Based on previously study, we want to study the effect of allacin on gene expression of HPV18-E6, hTERT and P53 of Hela cell in vitro. The purpose of this study is to provide a new way to prevent and cure the cervical cancer in clinic.
Methods
1 Cell culture: Hela cells freezed in liguid nitrogen were
revived in routine method, and inoculated in RPMI-1640 blended with 10% foetus cattle serum, cultured in culture bottle. The bottle was put in the incubator with the condition of 37℃、5%CO2. The growth of cells was observed every day. 70% of the cells confluence was digested with EDTA and then was made into cell suspension.
2 The growth suppression of cell was estimated by MTT: adjusted deuterium of cells to 3×104 / ml, mixed them completely and inoculated them to cultivation Plate of 96–pore, 200μl in every pore. Cells were put to incubator and cultured to logarithm phrase. Then they were got out , the superior liquor was got rid of and 200μl culture medium including drug was added. The concentration of drug are 25mg/L、 50 mg/L、 100 mg/L. Then they were put back to incubator, watched everyday. 20μl MTT was added in every pore after cultured 24、48、72 hours, cultured in incubator again for 4 hours. Then, all liquor was got rid of. 200μl di–methyl–Sulfoxide was added, shocked for 8 minutes. And then the number of A and calculated the inhibition rate were measured. The sequences were done for 3 times in the same way.
3 Observation by inverted microscope. Hela cell suspensions were seeded in 24~well culture plates at a density of 3×104 cells/ml (1ml/well) in culture medium. Experimental treatment of cells was same as above. 24 and 72 h later, cells were observed and shot by inverted microscope. Observation after Giemsa staining by light microscope. Hela cell suspensions were seeded in 24-well culture plates at a density of 3 x 104 cells/ml (1ml/well) in culture
medium with a small piece of cover glass in each well. Experimental treatment of cells was same as above. 72 h later, the glass was taken out and stained with Giemsa stain for 10 to 15 min. Cells were observed by light microscope.
4 Observation by electron microscope. Hela cell suspensions were seeded in 100ml culture flasks. Experimental treatment of cells was same as above. 72 h later, the cells were harvested by treatment with EDTA(0.02%) and fixed by glutaral(4%) at 4 ℃. After desiccation, penetration, embedding, section and staining, cells were observed by electron microscope (JEM-1230, Japan).
5 Assayed by TUNEL Test：Experimental treatment of cells was same as above. The cells were treated according to instruction of TUNEL Test Kit. The cells whose nucleuses were stained by brown were apoptosis cells, and the blue are negative. 300 cells were counted in every section, and then calculated the apoptosis rate of cells.
6 With PCR and RT-PCR, the expression change of HPV18E6 mRNA, p53 mRNA as well as hTERT mRNA were studied in the process of Hela cell apoptosis in vitro.
Results
1. Observation after Giemsa staining by light microscope：After Giemsa staining, cytoplasm of Hela cells in untreated group was pink and nucleus the number of which was two or three were royal blue. Cell membrane and nucleus were integrated. More karyokinesis and few chromatin margi
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Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumor suppression. Cell, 1993, 75: 817~825
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Pater MM, Pater A. Human papillomavirus types 16 and 18 sequences in carcinoma cell lines of the cervix. Virology, 1985, 145: 313~318
Cooper K, Herrington CS, Stickland JE, et al. Episomal and integrated human papillomavirus in cervical neoplasia shown by non–isotopic in situ hybridization J. Clin. Pathol, 1991, 44: 990~996
Scheffner M, Werness BA, Huibregtse JM, et al. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell, 1990, 63: 1129~1136
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Song S, Pitot HC, Lambert PG, et al. The human papillomavirus type 16 E6 gene alone sufficient to induce carcinomas in transgenic animals. J Viro, 1999, 73: 5787~5793
Park C S, Joo I S, Song S Y, et al. An immunohistochemical analysis of heat shock protein 70, p53 and estrogen receptor status in carcinoma of the uterine cervix. Gynecol Oncol, 1999, 74: 53~60
Skomedal H, Kristensen G B, Lie A K, et al. Aberrant expression of the cell cycle associated protein P53, MDM2, P21, P27, Cdk4, Cyclin D1, RB and EGFR in Cervical Carcinomas. Gynecol Oncol, 1999, 73: 233~228
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