整合素β_1阳性卵巢癌细胞具干细胞样属性和间质属性分析

Analysis on stem cell- like property and interstitial property of integrin β_1 positive ovarian cancer cells

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 positive cells in human ovarian cancer SKOV3 cell line and primary ovarian cancer cell line, identify whether they possess biological characteristics of tumor stem ceils. Methods： After successfully isolating ovarian cancer cells from ascites of patients with clinical ovarian cancer, flow cytometry was performed to detect the positive rates of integrin β1 and stem cell marker CD133 in ovarian cancer SKOV3 cells and primary ovarian cancer cells. Integrin β1 positive cells and integrin β1 negative cells were obtained, then qRT - PCR was performed to detect the expressions of ovarian caner stem cell - related genes （ CD44, CD133, AL- DH1, OCT） and epithelial- mesenchymal transition molecules （E- cadherin, N- eadherin, Vimentine, MMP- 2, MMP- 9）, the potentials of stem cells in the two groups were observed by suspension sphere formation test. Limiting dilution transplantation assay was used to compare the tumorigenic ability, self- renewal ability and differentiation ability between integrin β1 positive ceils and integrin [31 negative cells. Results：A few CD133 （ ＋ ） cells were detected in SKOV3 cells, the ratio was （0. 91 ＋0. 12）% , and the ratio of CD133 （ ＋ ） cells in primary ceils isolated from ascites was （2. 38 ±0. 34 ）%. The ratio of integrin β1 positive ceils in ovarian cancer SKOV3 cell line was （ 1.95 ±0. 24） %, and the ratio in primary cells was （3.78 ±0. 28） %. Compared to integrin β1 negative cells,integrin β1 positive cells expressed higher levels of stem cell genes （ CD133, CD44, ALDH1, OCT4 ） （ P 〈 0. 05 ） and lower level of epithelial marker E - cadherin （ P 〈 0. 05 ） and higher levels of epithelial - mesenchymal transition molecules （ N - cadherin, Vimentine, MMP - 2, MMP - 9 ） （ P 〈 0. 05 ）. Suspension sphere formation test showed that in ovarian cancer SKOV3 cells and primary cells, integrin β1 positive cells formed more spheres compared with integrin β1 negative cells, and the sphere volume was higher than that of integrin β1 negative cells （P 〈 0. 05）. In ovarian cancer SKOV3 cells and primary cells, 102 integrin β1 positive cells could generate tumors; tumor generation ratio was 1/5 and 2/5, respectively; tumor generation times were 64 days and 54 days, respectively. While at least 104 integrin β1 negative cells of ovarian cancer SKOV3 cell line could generate tumors, at least 103 integrin β1 negative cells of primary cells could generate tumors, both the tumor generation ratios were 1/5 ; tumor generation times were 78 days and 68 days, respectively; the tumor formation capability of integrin β1 positive cells was at least 100 times more than that of integrin β1 negative cells. Condusion：Integrin β1 positive cells highly express epithelial -mesenchymal transition molecules and ovarian caner stem cell - related genes, possess the abilities of self - differentiation, self - renewal, and tumor generation in vivo. Integrin β1 positive phenotype can be considered as a marker to isolate ovarian cancer stem cells.