中国癌症杂志
中國癌癥雜誌
중국암증잡지
CHINA ONCOLOGY
2013年
3期
161-166
,共6页
林凤娟%杨小利%郭雅洁%邵志敏%欧周罗
林鳳娟%楊小利%郭雅潔%邵誌敏%歐週囉
림봉연%양소리%곽아길%소지민%구주라
乳腺肿瘤%趋化因子%CCL28%基因表达%增殖
乳腺腫瘤%趨化因子%CCL28%基因錶達%增殖
유선종류%추화인자%CCL28%기인표체%증식
Breast cancer%Chemokine%CCL28%Gene expression%Proliferation
背景与目的:CCL28,又称为黏膜相关上皮趋化因子(mucosa-associated epithelia chemokine,MEC),是趋化因子CC家族中的一员.研究表明,CCL28与肿瘤发生、发展有关,但对其在乳腺癌中的功能还知之甚少.本文旨在探讨CCL28过表达在体外对乳腺癌细胞MDA-MB-231增殖的影响.方法:构建CCL28过表达载体pBabe-CCL28表达质粒,将重组的pBabe-CCL28基因和空载体pBabe转染细胞Phoenix,利用逆转录病毒感染乳腺癌细胞MDA-MB-231,通过嘌呤霉素药物筛选获得稳定表达CCL28的MDA-MB-231/CCL28细胞系和感染空载体的MDA-MB-231/vector细胞系,利用半定量RT-PCR、实时荧光定量PCR及蛋白质印迹法(Western blot)检测CCL28的基因表达和蛋白质水平;利用CCK8、软琼脂克隆培养及流式细胞术检测CCL28过表达后细胞增殖、克隆形成能力及凋亡的变化;利用Western blot检测CCL28过表达后凋亡相关蛋白的变化.结果:成功建立了稳定表达CCL28的细胞系MDA-MB-231/CCL28,与空载体对照组相比,pBabe-CCL28质粒感染入细胞后CCL28基因表达水平明显增高,说明CCL28 cDNA重组质粒构建成功.CCK8及软琼脂克隆实验结果显示,MDA-MB-231/CCL28的增殖能力明显高于MDA-MB-231/vector,差异有统计学意义(P<0.05).流式细胞术结果显示,MDA-MB-231/CCL28细胞凋亡比例减少,差异有统计学意义(P<0.05).Western blot结果显示,抗凋亡蛋白Bcl-2的表达增加.结论:CCL28过表达可以显著促进人乳腺癌细胞MDA-MB-231的增殖能力,抑制乳腺癌细胞凋亡,后者可能与Bcl-2上调有关.CCL28有望成为临床治疗乳腺癌的潜在基因靶点.
揹景與目的:CCL28,又稱為黏膜相關上皮趨化因子(mucosa-associated epithelia chemokine,MEC),是趨化因子CC傢族中的一員.研究錶明,CCL28與腫瘤髮生、髮展有關,但對其在乳腺癌中的功能還知之甚少.本文旨在探討CCL28過錶達在體外對乳腺癌細胞MDA-MB-231增殖的影響.方法:構建CCL28過錶達載體pBabe-CCL28錶達質粒,將重組的pBabe-CCL28基因和空載體pBabe轉染細胞Phoenix,利用逆轉錄病毒感染乳腺癌細胞MDA-MB-231,通過嘌呤黴素藥物篩選穫得穩定錶達CCL28的MDA-MB-231/CCL28細胞繫和感染空載體的MDA-MB-231/vector細胞繫,利用半定量RT-PCR、實時熒光定量PCR及蛋白質印跡法(Western blot)檢測CCL28的基因錶達和蛋白質水平;利用CCK8、軟瓊脂剋隆培養及流式細胞術檢測CCL28過錶達後細胞增殖、剋隆形成能力及凋亡的變化;利用Western blot檢測CCL28過錶達後凋亡相關蛋白的變化.結果:成功建立瞭穩定錶達CCL28的細胞繫MDA-MB-231/CCL28,與空載體對照組相比,pBabe-CCL28質粒感染入細胞後CCL28基因錶達水平明顯增高,說明CCL28 cDNA重組質粒構建成功.CCK8及軟瓊脂剋隆實驗結果顯示,MDA-MB-231/CCL28的增殖能力明顯高于MDA-MB-231/vector,差異有統計學意義(P<0.05).流式細胞術結果顯示,MDA-MB-231/CCL28細胞凋亡比例減少,差異有統計學意義(P<0.05).Western blot結果顯示,抗凋亡蛋白Bcl-2的錶達增加.結論:CCL28過錶達可以顯著促進人乳腺癌細胞MDA-MB-231的增殖能力,抑製乳腺癌細胞凋亡,後者可能與Bcl-2上調有關.CCL28有望成為臨床治療乳腺癌的潛在基因靶點.
배경여목적:CCL28,우칭위점막상관상피추화인자(mucosa-associated epithelia chemokine,MEC),시추화인자CC가족중적일원.연구표명,CCL28여종류발생、발전유관,단대기재유선암중적공능환지지심소.본문지재탐토CCL28과표체재체외대유선암세포MDA-MB-231증식적영향.방법:구건CCL28과표체재체pBabe-CCL28표체질립,장중조적pBabe-CCL28기인화공재체pBabe전염세포Phoenix,이용역전록병독감염유선암세포MDA-MB-231,통과표령매소약물사선획득은정표체CCL28적MDA-MB-231/CCL28세포계화감염공재체적MDA-MB-231/vector세포계,이용반정량RT-PCR、실시형광정량PCR급단백질인적법(Western blot)검측CCL28적기인표체화단백질수평;이용CCK8、연경지극륭배양급류식세포술검측CCL28과표체후세포증식、극륭형성능력급조망적변화;이용Western blot검측CCL28과표체후조망상관단백적변화.결과:성공건립료은정표체CCL28적세포계MDA-MB-231/CCL28,여공재체대조조상비,pBabe-CCL28질립감염입세포후CCL28기인표체수평명현증고,설명CCL28 cDNA중조질립구건성공.CCK8급연경지극륭실험결과현시,MDA-MB-231/CCL28적증식능력명현고우MDA-MB-231/vector,차이유통계학의의(P<0.05).류식세포술결과현시,MDA-MB-231/CCL28세포조망비례감소,차이유통계학의의(P<0.05).Western blot결과현시,항조망단백Bcl-2적표체증가.결론:CCL28과표체가이현저촉진인유선암세포MDA-MB-231적증식능력,억제유선암세포조망,후자가능여Bcl-2상조유관.CCL28유망성위림상치료유선암적잠재기인파점.
@@@@Background and purpose: CCL28, also known as mucosa-associated epithelial chemokine (MEC), is a member of the CC subfamily. Studies have shown that CCL28 is associated with tumor progress. However, little is known about its function in breast cancer. In this study, we construct the expression vector of cDNA encoding CCL28 gene and observe its effects on cell growth ability of human breast cancer cell line MDA-MB-231 in vitro. Methods: The pBabe-CCL28 expression vector was constructed, then transfected into Phoenix cells and used to infect cultured breast cancer cells MDA-MB-231 by using retroviruses infection method. The positive clones were selected with puromycin to establish stable transfectants MDA-MB-231/CCL28. Control cell lines were generated by infection with viruses containing empty vector by following the same protocol. CCL28 mRNA and protein in MDA-MB-231/CCL28 were determined by semi-quantitative RT-PCR, realtime PCR and western blot respectively. Cell proliferation was tested by cell counting kit 8 (CCK8). Cell apoptosis percentage was observed by flow cytometry. The ability of colony formation was tested by soft agar. The expression of apoptosis associated protein was measured by Western blot. Results: The cell line MDA-MB-231/CCL28 stabling expresses chemokine CCL28 was successfully constructed. In the MDA-MB-231/CCL28 cells, cell proliferation was increased, and cell apoptosis percentage was decreased. Western blot showed that the expression of anti-apoptotic protein Bcl-2 was decreased. Conclusion: Our data provides strong evidence that overexpression of CCL28 can promote the proliferative ability of MDA-MB-231 and inhibit the cell apoptosis, which may be regulated by Bcl-2. Therefore, CCL28 could be a important target for the treatment of breast cancer.