食蟹猴耳部成纤维细胞体外培养和增殖的初步研究
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摘要
本论文选取了非人灵长类动物——食蟹猴为研究对象,采用组织块贴壁培养法,构建了食蟹猴耳部组织成纤维细胞的体外培养体系,并对其生长特性进行了研究。在建立培养体系的基础上,运用MTT比色法分析了不同浓度表皮生长因子(EGF)、胰岛素(Insulin)和胰岛素样生长因子-I(IGF-I)对5%和10%两种血清浓度下培养细胞增殖能力的影响。
研究结果如下:
1、采用组织块培养法可以获得食蟹猴耳部原代培养物。
2、用0.25%胰蛋白酶+0.02%EDTA消化液消化细胞、用含有10%FBS的DMEM对细胞进行培养,细胞在体外传至第25代。3～5次传代后,培养细胞逐渐纯化,为典型的成纤维型细胞,绝大部分呈梭形或不规则三角形。细胞生长时呈放射状、火焰状或旋涡状走势。
3、用含10%FBS和10%DMSO的DMEM为冷冻液,采取手工冷冻法对细胞进行冷冻保存,冻存浓度为5×10~6个/ml。通过台盼蓝染色法对不同代数细胞冻存前后活率进行测定,结果发现虽然复苏后存活率有所下降,但均保持在90%以上,传代后细胞形态和生长速度没有明显变化。
4、绘制的细胞生长曲线呈“S”型,细胞生长经历了潜伏期、指数生长期和停滞期,符合体外细胞生长规律。
5、对第10、15、20和25代细胞的染色体倍性进行分析,发现二倍体染色体数为2n=42。虽然随着传代次数的增加,二倍体细胞所占比例有所下降,但仍在80%以上,且四代之间无显著差异(P＞0.05)。
6、当培养液中血清浓度为5%时,分别添加20～80 ng/ml的EGF、1、5μg/ml的胰岛素、50～100 ng/ml浓度的IGF-I可显著促进细胞增殖(P＜0.05)。
7、当培养液中血清浓度为10%时,分别添加10～40 ng/ml的EGF、10、15μg/ml的胰岛素、25～100 ng/ml浓度范围内的IGF-I可显著促进细胞增殖(P＜0.05)。
Macaca fascicularis was selected as experiment material in this study.By the means of tissue block culture,we aimed to establish an isolation method and culture system for Macaca fascicularis ear fibroblast cells in vitro.The biological characteristics of the ear fibroblast cells were also studied.In addition,the effects of supplementation of different concentration of EGF, insulin、IGF-I for cell proliferation of Macaca fascicularis ear fibroblast cells in the 5%and 10%FBS concentration were also evaluated by colorimetric MTT assay.
The results showed that:
1、Primary culture of Macaca fascicularis ear fibroblast cells could be obtained through tissue block culture successfully.
2、Using 0.25%trypsin+0.02%EDTA as digested medium and DMEM containing 10%FBS as culture medium for subculture of medium,and the cells were passaged to the 25~(th)passage.The pured fibroblast cells could be obtained by passed 3～5 subcultures.The cultured cells were typical fibroblast morpha, which grooved as irregular triangle or shuttle,appearing as radioactive blaze and helix in shape.
3、The fibroblast cells were frozen in DMEM containing 10%FBS and 10%DMSO as freezing medium with the manual methods,and the cell freezing denisity was approximately 5×10~6 cell/ml.The cells survival rate of the cells was observed by trypan blue staining method.The results showed that the cells might be affected by many experiment factors during cryopreservation,but there was more than 90%of cells survived after frozen and thawed.The cells before and after freezing had not showed obvious changes in cell morphology and growth speed.
4、The growth curve appeared in "S" shape.The cells understood latent phase,logarithmic growth phase and stagnate phase.The result accorded with the cells growth regularity in vitro.
5、The number of chromosome is 2n=42 in Macaca fascicularis on the 10~(th)、15~(th)、20~(th)and 25~(th)passages cells.Results of chromosome ploidy analysis indicated that cells are diploid,and there had no significant difference on the ratio of diploid cells among these 4 passages(P＞0.05).
6、when the serum concentration was 5%in DMEM,the addition of EGF (20～80 ng/ml)、insulin(1～5μg/ml)and IGF-I(50～100 ng/ml)had significant promoting effects on the growth of the cells respectively(P＜0.05).
7、when the serum concentration was 10%in DMEM,the addition of EGF (10～40 ng/ml)、insulin(10～15μg/ml)and IGF-I(25～100 ng/ml)had significant promoting effects on the growth of the cells respectively(P＜0.05).
研究结果如下:
1、采用组织块培养法可以获得食蟹猴耳部原代培养物。
2、用0.25%胰蛋白酶+0.02%EDTA消化液消化细胞、用含有10%FBS的DMEM对细胞进行培养,细胞在体外传至第25代。3～5次传代后,培养细胞逐渐纯化,为典型的成纤维型细胞,绝大部分呈梭形或不规则三角形。细胞生长时呈放射状、火焰状或旋涡状走势。
3、用含10%FBS和10%DMSO的DMEM为冷冻液,采取手工冷冻法对细胞进行冷冻保存,冻存浓度为5×10~6个/ml。通过台盼蓝染色法对不同代数细胞冻存前后活率进行测定,结果发现虽然复苏后存活率有所下降,但均保持在90%以上,传代后细胞形态和生长速度没有明显变化。
4、绘制的细胞生长曲线呈“S”型,细胞生长经历了潜伏期、指数生长期和停滞期,符合体外细胞生长规律。
5、对第10、15、20和25代细胞的染色体倍性进行分析,发现二倍体染色体数为2n=42。虽然随着传代次数的增加,二倍体细胞所占比例有所下降,但仍在80%以上,且四代之间无显著差异(P＞0.05)。
6、当培养液中血清浓度为5%时,分别添加20～80 ng/ml的EGF、1、5μg/ml的胰岛素、50～100 ng/ml浓度的IGF-I可显著促进细胞增殖(P＜0.05)。
7、当培养液中血清浓度为10%时,分别添加10～40 ng/ml的EGF、10、15μg/ml的胰岛素、25～100 ng/ml浓度范围内的IGF-I可显著促进细胞增殖(P＜0.05)。
Macaca fascicularis was selected as experiment material in this study.By the means of tissue block culture,we aimed to establish an isolation method and culture system for Macaca fascicularis ear fibroblast cells in vitro.The biological characteristics of the ear fibroblast cells were also studied.In addition,the effects of supplementation of different concentration of EGF, insulin、IGF-I for cell proliferation of Macaca fascicularis ear fibroblast cells in the 5%and 10%FBS concentration were also evaluated by colorimetric MTT assay.
The results showed that:
1、Primary culture of Macaca fascicularis ear fibroblast cells could be obtained through tissue block culture successfully.
2、Using 0.25%trypsin+0.02%EDTA as digested medium and DMEM containing 10%FBS as culture medium for subculture of medium,and the cells were passaged to the 25~(th)passage.The pured fibroblast cells could be obtained by passed 3～5 subcultures.The cultured cells were typical fibroblast morpha, which grooved as irregular triangle or shuttle,appearing as radioactive blaze and helix in shape.
3、The fibroblast cells were frozen in DMEM containing 10%FBS and 10%DMSO as freezing medium with the manual methods,and the cell freezing denisity was approximately 5×10~6 cell/ml.The cells survival rate of the cells was observed by trypan blue staining method.The results showed that the cells might be affected by many experiment factors during cryopreservation,but there was more than 90%of cells survived after frozen and thawed.The cells before and after freezing had not showed obvious changes in cell morphology and growth speed.
4、The growth curve appeared in "S" shape.The cells understood latent phase,logarithmic growth phase and stagnate phase.The result accorded with the cells growth regularity in vitro.
5、The number of chromosome is 2n=42 in Macaca fascicularis on the 10~(th)、15~(th)、20~(th)and 25~(th)passages cells.Results of chromosome ploidy analysis indicated that cells are diploid,and there had no significant difference on the ratio of diploid cells among these 4 passages(P＞0.05).
6、when the serum concentration was 5%in DMEM,the addition of EGF (20～80 ng/ml)、insulin(1～5μg/ml)and IGF-I(50～100 ng/ml)had significant promoting effects on the growth of the cells respectively(P＜0.05).
7、when the serum concentration was 10%in DMEM,the addition of EGF (10～40 ng/ml)、insulin(10～15μg/ml)and IGF-I(25～100 ng/ml)had significant promoting effects on the growth of the cells respectively(P＜0.05).
引文
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