雌、孕激素对体外培养及荷瘤裸鼠体内宫颈腺癌HeLa细胞生长影响的研究

发布时间：2018-06-28 07:27

本文选题：雌激素 + 孕激素　；参考：《南昌大学》2017年博士论文

【摘要】：目的用性激素E2、P或E2+P作用于体外培养及荷瘤裸鼠的Hela细胞,检测细胞增殖、细胞周期及侵袭迁移能力的变化情况;初步评价激素干预对体外培养及荷瘤裸鼠体内HeLa细胞生长的影响,为临床进行宫颈腺癌激素替代治疗提供基础实验依据。方法1.采用免疫组化方法检测体外培养的人宫颈腺癌HeLa细胞雌、孕激素受体的表达;2.不同浓度的E2、P和E2+P分别与HeLa细胞共培养,用CCK8检测各实验组培养后24小时、48小时及72小时Hela细胞的OD值,以观察其增殖情况;3.用Transwell板检测E2、P和E2+P作用48小时后体外培养Hela细胞的细胞迁移、侵袭能力的变化;4.用流氏细胞仪检测E2、P和E2+P与Hela细胞共培养48小时后细胞周期及调亡的变化;5.于裸鼠双侧后肢背部皮下注射Hela细胞悬混液,24小时后分别予E2、P和E2+P腹腔注射治疗,每日一次,建立Hela细胞肿瘤的荷瘤裸鼠,并继续用E2,P和E2+P分组治疗,观察肿瘤生长情况及裸鼠存活时间的差异;结果1.用免疫组化二步法检测Hela细胞Er、Pr表达,发现体外培养的Hela细胞Er、Pr表达强阳性;2.结果显示,E2、P和E2+P三组的10μmol/L、100μmol/L浓度对Hela细胞生长均有抑制作用,尤其是在培养48小时与72小时后对Hela细胞的抑制作用明显,与对照组相比差异有统计学意义(p0.05),且药物浓度更高的100μmol/L组对Hela细胞的抑制作用更强,而三种药物的其余浓度组(1μmol/L、0.1μmol/L、0.01μmol/L)对Hela细胞的生长影响不明显,与对照组相比无统计学意义(p0.05),因此选择对细胞抑制作用最强的100μmol/l浓度组进行后续实验;实验的各药物浓度组均未观察到其对hela细胞生长的促进作用;3.hela细胞加入激素培养48小时后,经流式细胞仪检测空白对照组、e2、p和e2+p组的凋亡率分别为:5.40±2.38%、20.1±2.20%、20.03±1.91%、24.40±2.40%,各组凋亡率增加,与对照组相比差异有统计学意义(p0.05),实验各组均未观察到细胞凋亡减少情况;细胞周期方面,实验观察到e2、p、e2+p组均表现为g0/g1期比例升高,s+g2/m期比例下降,与对照组相比,差异有统计学意义(p0.05)。4、在我们的实验药物浓度下(100μmol/l)培养hela细胞48小时,单独e2或p对hela细胞迁移、侵袭能力无明显影响,细胞迁移及侵袭数与对照组相比差异无统计学意义(p0.05),但e2+p联合使用对hela细胞迁移、侵袭能力减弱,迁移、侵袭细胞数与对照组相比有差异(p0.05);在我们的实验药物浓度下未观察到对细胞迁移、侵袭的促进作用。5.一周后裸鼠成功建立hela细胞荷瘤裸鼠模型,肿瘤接种24小时即分别开始予e2、p和e2+p治疗,空白对照组、e2组、p组及e2+p组成瘤率分别是:87.5%(14/16)、100%(15/15)、81.25%(13/16)、100%(16/16),各组间成瘤率无明显差异(p0.05)。实验裸鼠用药82天,空白对照组、e2组、p组及e2+p组的平均肿瘤体积分别是105.89mm3、450.17mm3、107.96mm3及168.94mm3,其中e2与对照组相比,肿瘤体积更大,有统计学意义(p0.05),各组裸鼠体重之间差异无统计学意义(p0.05);肿瘤体积最大者出自e2组,e2组的1号鼠有腹腔转移瘤(体重19.5g)、e2+p组的2号和7号鼠分别有头部及腹腔转移(二者体重分别为15g及9.9g),并且均在81天死亡,转移率分别为12.5%(1/8)及25%(2/8),其中e2组的转移瘤体积为171.5mm3,e2+p的转移瘤体积分别为1070mm3和600mm3,e2+p转移瘤体积明显更大。结论性激素e2、p及e2+p联合在一定浓度下对体外培养的hela细胞有抑制作用;适当溶度的e2+p可能降低体外培养的hela细胞的侵袭、迁移能力;在荷瘤裸鼠体内,一定浓度e2促进肿瘤生长,e2+p可能促进肿瘤细胞的早期转移。
[Abstract]:Objective to evaluate the effect of sex hormone E2, P or E2+P on Hela cells in vitro and tumor bearing nude mice, to detect the changes of cell proliferation, cell cycle and invasion and migration, and to evaluate the effect of hormone intervention on the growth of HeLa cells in vitro and in vivo in nude mice. Method 1. immunohistochemistry was used to detect the expression of estrogen and progesterone receptor in human cervical adenocarcinoma HeLa cells in vitro, and 2. different concentrations of E2, P and E2+P were co cultured with HeLa cells respectively. CCK8 was used to detect the OD value of Hela cells in 24 hours, 48 hours and 72 hours after culture in each experiment group, to observe the proliferation of HeLa cells, and 3. to detect E2 with Transwell plate. 48 hours after the action of P and E2+P, the cell migration and invasiveness of Hela cells were cultured in vitro. 4. the cell cycle and the change of apoptosis after 48 hours of co culture of E2, P and E2+P with Hela cells were detected by flow cytometer. 5. in the back of the hind limbs of nude mice, Hela cell suspension was injected subcutaneously, and the treatment of E2, P and E2+P intraperitoneal injection after 24 hours. Once a day, the tumor bearing nude mice of Hela cell tumor were established and the E2, P and E2+P groups were continued to be treated to observe the difference between the tumor growth and the survival time of nude mice. Results 1. the Er and Pr expression of Hela cells were detected by immunohistochemical two step method, and the Hela cells in vitro were found to be Er, Pr expression was strong positive; 2. the results showed that E2, P, and three groups of 10 micron, The concentration of 100 mu mol/L could inhibit the growth of Hela cells, especially the inhibition effect on Hela cells after 48 hours and 72 hours, compared with the control group, the difference was statistically significant (P0.05), and the higher concentration of 100 mu mol/L on the inhibition of Hela cells was stronger, while the remaining concentration group of the three drugs (1 mu mol/L, 0.1 mu). The effect of mol/L, 0.01 mu mol/L) on the growth of Hela cells was not obvious, and there was no significant difference compared with the control group (P0.05). Therefore, a follow-up experiment was carried out in the group of 100 mu mol/l which had the strongest inhibitory effect on the cells. All the concentration groups in the experiment did not observe the promotion effect on the growth of HeLa cells, and 3.hela cells added hormone for 48 hours. After the flow cytometry, the apoptosis rate of E2, P and e2+p groups were 5.40 + 2.38%, 20.1 + 2.20%, 20.03 + 1.91% and 24.40 + 2.40% respectively. The apoptosis rate of each group increased, and the difference was statistically significant (P0.05) compared with the control group (P0.05). The cell cycle aspect, the cell cycle aspect, the experiment observed E2, P, e2+p. The proportion of g0/g1 stage and s+g2/m phase decreased in all groups. Compared with the control group, the difference was statistically significant (P0.05).4. In our experimental drug concentration (100 mu mol/l), HeLa cells were cultured for 48 hours, and E2 or P alone had no significant influence on the migration of HeLa cells, and the number of cell migration and invasion was not statistically significant compared with the control group. Learning significance (P0.05), but e2+p combined use of HeLa cell migration, invasion ability weakened, migration, the number of invasive cells compared with the control group is different (P0.05); in our experimental drug concentration, no observation of cell migration, invasion of.5. a week after the establishment of nude mice to establish a tumor tumor nude mice model, tumor inoculation for 24 hours that is. Do not start with E2, P and e2+p treatment. The tumor rate in blank control group, E2 group, P group and e2+p was 87.5% (14/16), 100% (15/15), 81.25% (13/16), 100% (16/16), and there was no significant difference between each group (P0.05). The average tumor volume of the experimental nude mice was 82 days. 168.94mm3, compared with the control group, the tumor volume was larger than the control group (P0.05), and there was no significant difference between the body weight of the nude mice (P0.05). The largest tumor volume was from the E2 group, and the 1 mice in group E2 had abdominal metastasis (weight 19.5g), and the 2 and 7 mice of e2+p group had head and abdominal metastasis respectively (two of them were 15g respectively. The metastatic rate was 12.5% (1/8) and 25% (2/8) at 81 days. The volume of metastatic tumor in group E2 was 171.5mm3, and the volume of metastatic tumor of e2+p was 1070mm3 and 600mm3, and the volume of e2+p metastatic tumor was significantly larger. Conclusion sex hormone E2, P and e2+p were inhibited under one fixed concentration. The degree of e2+p may reduce the invasion and migration of HeLa cells in vitro, and in the tumor bearing nude mice, a certain concentration of E2 promotes the growth of the tumor, and e2+p may promote the early metastasis of tumor cells.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.33

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