Wei Zhentong, Wang Yishu, Yu Xiaowei, Zhang Songling
Department of Obstetrics and Gynecology, the First Hospital of Jilin University, Changchun 130021, China.
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Zhonghua Fu Chan Ke Za Zhi. 2015 Jun;50(6):452-7.
To isolate side population (SP) cells from an established ovarian cancer (OC) cell line, characterize these cells, and examine their drug resistance.
SP and non-SP (NSP) cells were isolated by fluorescence-activated cell sorting (FACS), and cultured in differential conditions, then detected their SP ratio to compare their capability of differentiation and self-renewal. Moreover, SP and NSP cell tumorigenesis was examined by subcutaneous and intraperitoneal injection of these cells to nonobes ediabetic (NOD)-severe combined immundeficient (SCID) mice. Drug resistance to cisplatin was examined by cell counting kit-8 (CCK-8).
SP cells could be isolated stablly and insistently. There was (4.81 ± 0.43)% of SP cells in the established OC cell line and (4.89 ± 0.33)% of SP cells after cultured the isolated SP cells in differentiation condition, and there was no significant different between these two quantities (P > 0.05). However, after cultured the NSP cells, there was only (0.10 ± 0.03)% of SP cells which was significantly lower than that contained in the OC cell line (P < 0.01). In the tumorigenesis assay 1.0 × 10(3) SP cells were injected subcutaneously and formed the xenografted tumors in 6 weeks (3/3), and 1.0 × 10(4) NSP cells were injected subcutaneously and did not form xenografted tumors in 12 weeks (0). The tumorigenic capability of SP cells was higher than that of NSP cells (P < 0.01). Both the original and the xenografted tumors were low differentiated serous cystadenocarcinomas and expressed the ovarian serous cystadenocarcinomas CA125 marker after stained by HE and immunohistochemistry. Simultaneously, the SP cells were also capable to form tumors as shown by intraperitoneal injection. In the drug resistance assay shown that the 50% inhibitory concentration (IC50) of the SP and NSP cells were respectively (2.33 ± 0.14) µg/ml and (1.60 ± 0.04) µg/ml (P < 0.05). After treated the unsorted OC cells with cisplatin, the quantity of SP cells increased to (40.10 ± 4.22)% and there was significant difference, when compared to the untreated cells which was (4.81 ± 0.43)% (P < 0.01). The SP cells survival rate was (58.7 ± 3.3)% when treated with cisplatin at its IC50 dose, and the rate decreased to (7.2 ± 1.3)% (P < 0.01) when verapamil was present.
The SP cells could be isolated from the established OC cell line. They had the capacities of self-renewal, differentiation, and tumorigenesis, and the new tumor demonstrated the original tumor's phenotype. The SP cells also had stem cells' biological characteristics and is resistant to cisplatin.
从已建立的卵巢癌细胞系中分离出侧群（SP）细胞，对这些细胞进行特性分析，并检测其耐药性。
通过荧光激活细胞分选（FACS）分离出SP和非SP（NSP）细胞，并在不同条件下培养，然后检测它们的SP比例，以比较其分化和自我更新能力。此外，通过将这些细胞皮下和腹腔注射到非肥胖糖尿病（NOD）-重症联合免疫缺陷（SCID）小鼠体内来检测SP和NSP细胞的致瘤性。通过细胞计数试剂盒-8（CCK-8）检测对顺铂的耐药性。
SP细胞能够稳定且持续地分离出来。在已建立的卵巢癌细胞系中，SP细胞占（4.81±0.43）%，将分离出的SP细胞在分化条件下培养后，SP细胞占（4.89±0.33）%，这两个比例之间无显著差异（P>0.05）。然而，培养NSP细胞后，仅出现（0.10±0.03）%的SP细胞，显著低于卵巢癌细胞系中的比例（P<0.01）。在致瘤性实验中，皮下注射1.0×10³个SP细胞，6周内形成异种移植瘤（3/3），皮下注射1.0×10⁴个NSP细胞，12周内未形成异种移植瘤（0）。SP细胞的致瘤能力高于NSP细胞（P<0.01）。原代肿瘤和异种移植瘤均为低分化浆液性囊腺癌，经苏木精-伊红（HE）染色和免疫组织化学染色后均表达卵巢浆液性囊腺癌CA125标志物。同时，腹腔注射显示SP细胞也能够形成肿瘤。在耐药性实验中，SP和NSP细胞的50%抑制浓度（IC50）分别为（2.33±0.14）μg/ml和（1.60±0.04）μg/ml（P<0.05）。用顺铂处理未分选的卵巢癌细胞后，SP细胞数量增加至（40.10±4.22）%，与未处理细胞的（4.81±0.43）%相比有显著差异（P<0.01）。当用IC50剂量的顺铂处理时，SP细胞存活率为（58.7±3.3）%，当存在维拉帕米时，该比例降至（7.2±1.3）%（P<0.01）。
可以从已建立的卵巢癌细胞系中分离出SP细胞。它们具有自我更新、分化和致瘤能力，新形成的肿瘤表现出原肿瘤的表型。SP细胞还具有干细胞的生物学特性且对顺铂耐药。
