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目的:检测和分选人卵巢癌细胞系SKOV3及原代卵巢癌细胞中整合素β1(ITGβ1)阳性细胞,鉴定其是否具有肿瘤干细胞生物学特性。方法:从临床卵巢癌患者腹水中成功分离卵巢癌细胞后,采用流式细胞技术检测SKOV3及原代卵巢癌细胞中ITGβ1和干细胞标志CD133的阳性率;流式分选得到ITGβ1(+)和ITGβ1(-)两群细胞后,采用qRT-PCR比较卵巢癌干细胞相关基因(CD44、CD133、ALDH1、OCT)及上皮间质化(EMT)分子(E-cadherin、N-cadherin、Vimentine、MMP2、MMP9)表达情况,采用悬浮成球试验观察两者干细胞潜能。最后通过免疫缺陷小鼠体内的有限稀释成瘤试验对比ITGβ1(+)/ITGβ1(-)细胞的成瘤能力、自我更新和自我分化能力。结果:SKOV3细胞中可检测到少量的CD133(+)细胞,其比率为(0.91±0.12)%,腹水原代细胞中CD133(+)比率为(2.38±0.34)%;ITGβ1检测SKOV3中ITGβ1(+)比率为(1.95±0.24)%,原代卵巢癌细胞中含量为(3.78±0.28)%;流式分选得到的ITGβ1(+)细胞较ITGβ1(-)细胞表达更高的干细胞基因(CD133、CD44、ALDH1、OCT4)(P<0.05)、更低的上皮标志E-cadherin(P<0.05)和更高的间质标志(N-cadherin、Vimentine、MMP2、MMP9)(P<0.05);悬浮成球试验结果显示:SKOV3细胞和原代卵巢癌细胞中ITGβ1(+)细胞较ITGβ1(-)细胞成球数量明显增多,球体体积也明显大于ITGβ1(-)细胞(P<0.05);体内成瘤试验结果显示:在SKOV3细胞系或原代卵巢癌细胞中102个ITGβ1(+)细胞即可成瘤,成瘤比率分别为1/5和2/5,成瘤时间分别为64天和54天;而至少需要104个ITGβ1(-)细胞SKOV3细胞系才有成瘤现象,至少需要103个ITGβ(-)细胞原代卵巢癌细胞才有成瘤现象,成瘤比率均为1/5,成瘤时间分别为78天和68天;ITGβ1(+)细胞的成瘤能力至少为ITGβ1(-)细胞的100倍。结论:卵巢癌细胞中ITGβ1(+)细胞高表达间质属性和干细胞基因,具备自我分化、自我更新和体内成瘤能力,ITGβ1(+)表型可考虑作为分选卵巢癌干细胞的新方法。
OBJECTIVE: To detect and sort integrin β1 (ITGβ1) positive cells in human ovarian cancer cell line SKOV3 and primary ovarian cancer cells, and to determine whether they possess tumor stem cell biological characteristics. Methods: The positive rates of ITGβ1 and stem cell marker CD133 in SKOV3 and primary ovarian cancer cells were detected by flow cytometry after successfully separating ovarian cancer cells from ascites in patients with clinical ovarian cancer. Flow cytometry (FCM) showed that ITGβ1 (+) and ITGβ1 QRT-PCR was used to compare the expression of CD44, CD133, ALDH1, OCT and E-cadherin (Vimentin, MMP2, MMP9 ) Expression, the use of suspended ball test to observe the potential of both stem cells. Finally, the ability of tumorigenesis, self-renewal and self-differentiation of ITGβ1 (+) / ITGβ1 (-) cells was compared with the limited dilution tumorigenesis test in immunodeficient mice. Results: The percentage of CD133 (+) cells in SKOV3 cells was (0.91 ± 0.12)% and that in primary ascites cells was (2.38 ± 0.34)%, and the number of ITGβ1 (1.95 ± 0.24)%, and (3.78 ± 0.28)% in primary ovarian cancer cells. Flow cytometry showed that ITGβ1 (+) cells expressed more stem cell genes than ITGβ1 (-) cells (P <0.05), lower epithelial marker E-cadherin (P <0.05) and higher interstitial markers (Vimentine, MMP2 and MMP9) The number of spheroids of ITGβ1 (+) cells in SKOV3 cells and primary ovarian cancer cells was significantly larger than that in ITGβ1 (-) cells, and the volume of spheroids was also significantly larger than that of ITGβ1 (-) cells (P <0.05). In vivo tumorigenicity test results showed that 102 ITGβ1 (+) cells could form tumors in SKOV3 cell line or primary ovarian cancer cells with tumorigenic rates of 1/5 and 2/5 respectively and tumorigenic time of 64 days And at least 54 days. At least 104 ITGβ1 (-) cells in SKOV3 cell line had tumorigenicity, at least 103 ITGβ (-) cells in primary ovarian cancer cells had tumorigenicity, the tumorigenic rate was 1 / 5, tumor time points 78 days and 68 days; tumorigenicity ITGβ1 (+) cells is at least ITGβ1 (-) 100 cell times. CONCLUSION: ITGβ1 (+) cells in ovarian cancer cells are highly expressed in mesenchymal properties and stem cell genes with self-differentiation, self-renewal and in vivo tumorigenicity. ITGβ1 (+) phenotype can be considered as a new method for the sorting of ovarian cancer stem cells.