人胆囊癌干细胞的分离、鉴定及其恶性生物学行为的调控研究
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摘要
第一部分体外无血清培养胆囊癌悬浮细胞球及其生物学特性研究
目的：通过体外培养扩增胆囊癌悬浮细胞球并分析其生物学特性,明确其中是否富含有肿瘤干细胞,并初步筛选其细胞表面标志物。
方法：将人原代胆囊癌组织和胆囊癌细胞系GBC-SD消化制成单细胞悬液,置入含有多种细胞因子的无血清培养液DMEM/F12中培养出肿瘤细胞克隆球；将培养出肿瘤细胞克隆球给予传代扩增；并将其置入含有血清的分化环境中贴壁培养,14天后实时定量RT-PCR检测干细胞标记物Oct-4和Nestin的表达；MTT法检测克隆球细胞和贴壁细胞的增殖能力和对化疗药的敏感性,并植入裸鼠皮下比较移植瘤形成能力；用流式细胞术法检测肿瘤干细胞标志物CD24、CD44、CD133在克隆球细胞和贴壁细胞中的表达。
结果：人原代胆囊癌细胞和GBC-SD细胞均可在干细胞培养环境中长出克隆球,并可以连续传代形成新的克隆球；克隆球细胞在含血清的环境中出现贴壁生长并分散开,克隆球渐变小最后消失,同时干细胞标记物Oct-4和Nestin的表达下调；与贴壁细胞相比,克隆球细胞在体外具有强的增殖、耐受吉西他滨和5-氟尿嘧啶和在裸鼠皮下形成新的移植瘤能力；经流式细胞术检测,CD133+细胞在克隆球细胞中的比例高于在贴壁细胞中的比例。
结论：人原代胆囊癌细胞和GBC-SD细胞中存在肿瘤干细胞,可以通过在干细胞培养条件下培养得到一定程度的扩增,CD133蛋白可能为其细胞表面标志物。
第二部分胆囊癌干细胞的分离及生物学特性鉴定
目的：用细胞表面标志物CD24、CD44、CD133分离出胆囊癌亚群细胞并进行肿瘤干细胞特性分析,鉴定出胆囊癌干细胞样细胞。
方法：将新鲜的人胆囊癌组织标本切成小块后置入裸鼠皮下建立裸鼠荷瘤模型,为下一步研究提供胆囊癌标本材料；将胆囊癌组织和GBC-SD细胞制成单细胞悬液后,标上抗人CD24-FITC、CD44-APC和CD133-PE流式抗体,流式细胞术检测并分离出CD24+、CD44+和CD133+细胞,进一步研究各亚群的生物学特性；将具有肿瘤干细胞特性的亚群标志物联合检测、分选亚群细胞及研究生物学特性包括在无血清培养环境中克隆形成球、对化疗药吉西他滨和5-氟尿嘧啶的敏感性、在NOD/SCID鼠体内形成异种移植瘤能力及体内自我更新、分化潜能。
结果：新鲜的人胆囊癌组织标本植入裸鼠皮下3-6月后大部分裸鼠成功长出移植瘤；经流式分析CD24+、CD44+和CD133+细胞在原代胆囊癌和GBC-SD细胞中的比例分别为：20.83%-35.72%和55.73%,78.19%～93.36%和96.78%,1.93%-3.43%和61.28%；经生物学特性研究,CD24+和CD24-细胞在形成克隆球、移植瘤形成能力上没有明显差别,差异性没有统计学意义(P>0.05),而CD44+和CD133+细胞在形成克隆球、移植瘤形成能力上均明显强于相应的阴性亚群(P<0.05)；流式进一步检测观察到CD44+CD133+细胞在原代肿瘤和GBC-SD细胞中的比例为2.53%和60.62%,且CD44+CD133+细胞较CD44+CD133-细胞具有更强的形成克隆球、耐受化疗和移植瘤形成能力；经流式分析和免疫组化检测CD44+CD133+细胞形成的移植瘤细胞中,既有CD44+和CD133+细胞,也含有CD44-和CD133-细胞。
结论：人胆囊癌CD44+CD133+细胞具有肿瘤干细胞的特性,其中富集有肿瘤干细胞,CD44和CD133可用来分离胆囊干细胞。
第三部分CD133特异siRNA对胆囊癌细胞生物学行为的影响及核转录因子Gli在胆囊癌细胞中的表达
目的：通过运用RNAi干扰技术沉默胆囊癌细胞株GBC-SD的CD133的表达,研究其对GBC-SD细胞的生物学行为的影响,研究核转录因子Gli在胆囊癌细胞中的表达。方法：将CD133特异性siRNA转染胆囊癌细胞株GBC-SD,并运用Western blot和流式细胞术分析并优化转染的效率和效果；在无血清环境中检测其克隆球形成能力,运用MTT分析其体外增殖和对5-氟尿嘧啶的耐受性,以细胞划痕实验检测其细胞迁徙能力；运用实时定量RT-PCR检测核转录因子Gli1、Gli2、Gli3在胆囊癌不同亚群中的表达。
结果：特异性siRNA转染后,经Western blot和流式细胞术分析,胆囊癌细胞株GBC-SD的CD133表达明显下调；转染CD133特异性siRNA组细胞较空白对照组和转染非特异性siRNA组细胞,其细胞克隆球形成能力、细胞增殖能力及细胞迁徙能力均明显下降(P<0.05),而对5-氟尿嘧啶的敏感性无明显差异(P>0.05)；核转录因子Gli1在胆囊癌CD44+CD133+细胞中表达上调。
结论：CD133与胆囊癌干细胞的增殖和细胞迁徙相关,可作为胆囊癌的治疗靶点之一；Glil可能参与胆囊癌干细胞生物学特性的调控。
Part I Culture of floating tumor spheres from human gallbladder carcinoma in serum-free media and study of the Biological characteristics
Objective To culture and expand the floating tumor spheres from human gallbladder carcinoma cells in vitro and study the biological characteristics of the sphere forming cells, and to determine whether cancer stem cells were contained in the gallbladder carcinoma and filter the cell surface markers preliminary.
Methods Human primary gallbladder carcinoma specimens and gallbladder cancer cell line GBC-SD were made into single cancer cells and cultured to grow into tumor spheres in serum-free DMEM/F12 media which contained some cell factors. The tumor spheres were passaged and expanded. Then differentiation analysis was performed by culturing in media containing serum and stem markers including Oct-4 and Nestin were detected using real time RT-PCR. MTT method was used to measure proliferation capacity and sensitivity to chemotherapeutic drugs, and tumorigenicity was measured by transplanted into the nude mice. Then the expression of CD24, CD44 and CD133 in spheres forming cells and adherent cells was tested using flow cytometry.
Results Both primary human gallbladder cancer cells and GBC-SD cells were found to grow into spheres in the serum-free environment and the sphere forming cells could reform new spheres. The floating tumor spheres became adherent and cells migrated from the spheres, and the spheres became smaller and disappeared in the differentiating environment. The expression of Oct-4 and Nestin was down-regulated in the adherent cells. Compared with the adherent cells, sphere forming cells showed the higher proliferation capacity, chemoresistance to gemcitabine and 5-fluorouracil and higher tumorigenicity. CD133+cells showed a higher proportion in sphere forming cells than in adherent cells.
Conclusion Human gllbladder carcinoma cells contain cancer stem cellst and the cancer stem cells could be expanded to some extent in stem cells environment, and CD 133 protein may be the cell surface marker of the cancer stem cells.
Part II Isolation and identification of cancer stem cells in human gallbladder carcinoma
Objective To isolate and identify the cancer stem cells in gallbladder carcinoma using CD24,CD44 and CD133 proteins.
Methods Fresh tissue samples of human gallbladder carcinoma were cut into smaller pieces and were transplanted into nude mice to established tumor model. Gallbladder carcinoma tissue and gallbladder cancer cell line GBC-SD were made into single cells and labeled anti-human CD24-FITC, CD44-APC and CD133-PE antibody. The CD24+,CD44+ and CD133+subset cells were isolated and the biological characteristics was studied. Then the multiple-marker isolation was performed and the biological characteristics of subset cells including sphere formation, sensitivity to chemotherapeutic drugs, tumorigenicity, self-renewal and differentiation potential were studied.
Results A majority of mouse tumor models were successfully established.The proportion of CD24+,CD44+ and CD133+ subset cells were 20.83%～35.72% and 55.73%, 78.19%～93.36% and 96.78%,1.93%～3.43% and 61.28%, in primary gallbladder carcinoma and cell line GBC-SD respectively. For biological characteristics, CD24+ and CD24 cells showed no significant difference in spheroid formation capacity or in tumorigenicity (P>0.05).While CD44+ and CD133+ gallbladder carcinoma cells showed higher spheroid formation capacity and tumorigenicity than their crresponding negative subsets (P<0.05). The proportion of CD44+ CD133+ subset was 2.53% and 60.62% in primary gallbladder carcinoma and cell line GBC-SD respectively. Compared to CD44+ CD 133, CD44+ CD 133+ subset cells showed higher spheroid formation capacity, chemoresistance and tumorigenicity (P<0.05). Both flow cytometry and immunohistochemistry showed that the transplantable tumors derived from CD44+ CD133+ cells contained not only CD44+ CD133+ cells,but CD44-and CD133-subsets.
Conclusion CD44+CD133cells exhibited cancer stem cells characteristics in human gallbladder carcinoma and the cancer stem cells were enriched in this subset cells.
PartⅢInfluence of CD133-specific siRNA on the biological behaviors of gallbladder cancer cells and expression of transcription factor Glis in gallbladder cancer cells
Objective To study the role of CD133 in gallbladder cancer cells by using CD133-specific siRNA to silence CD 133 expresion in GBC-SD.And to study the expression of of transcription factor Glis in gallbladder cancer cells.
Methods CD133-specific siRNA was transfected into gallbladder cancer cell line GBC-SD, and Western blot and flow cytometry were used to analyze and optimize the efficiency and effectiveness of transfection. Then tumor sphere formation capacity was detected in serum-free environment, and proliferation capacity and sensitivity to 5-Fu were tested using MTT method. A cell wounding assay was performed to analyze cell migration. Expression of transcription factors Glil, Gli2, Gli3 in CD44+CD133+cell and others subsets was detected using real time RT-PCR.
Results Expression of CD 133 was down-regulated after specific siRNA transfection. Compared with control cells, those transfected specific siRNA showed a lower tumor sphere formation, proliferation and cell migration capacity (P<0.05), while chemoresistance showed no significant change(P>0.05). Nuclear transcription factor Glil expression was upregulated in CD44+CD133+gallbladder cancer cells.
Conclusion CD 133 was associated with proliferation and migration of CSCs in GBC and Glil may be involved in the regulation of biological characteristics of gallbladder cancer stem cells.
目的：通过体外培养扩增胆囊癌悬浮细胞球并分析其生物学特性,明确其中是否富含有肿瘤干细胞,并初步筛选其细胞表面标志物。
方法：将人原代胆囊癌组织和胆囊癌细胞系GBC-SD消化制成单细胞悬液,置入含有多种细胞因子的无血清培养液DMEM/F12中培养出肿瘤细胞克隆球；将培养出肿瘤细胞克隆球给予传代扩增；并将其置入含有血清的分化环境中贴壁培养,14天后实时定量RT-PCR检测干细胞标记物Oct-4和Nestin的表达；MTT法检测克隆球细胞和贴壁细胞的增殖能力和对化疗药的敏感性,并植入裸鼠皮下比较移植瘤形成能力；用流式细胞术法检测肿瘤干细胞标志物CD24、CD44、CD133在克隆球细胞和贴壁细胞中的表达。
结果：人原代胆囊癌细胞和GBC-SD细胞均可在干细胞培养环境中长出克隆球,并可以连续传代形成新的克隆球；克隆球细胞在含血清的环境中出现贴壁生长并分散开,克隆球渐变小最后消失,同时干细胞标记物Oct-4和Nestin的表达下调；与贴壁细胞相比,克隆球细胞在体外具有强的增殖、耐受吉西他滨和5-氟尿嘧啶和在裸鼠皮下形成新的移植瘤能力；经流式细胞术检测,CD133+细胞在克隆球细胞中的比例高于在贴壁细胞中的比例。
结论：人原代胆囊癌细胞和GBC-SD细胞中存在肿瘤干细胞,可以通过在干细胞培养条件下培养得到一定程度的扩增,CD133蛋白可能为其细胞表面标志物。
第二部分胆囊癌干细胞的分离及生物学特性鉴定
目的：用细胞表面标志物CD24、CD44、CD133分离出胆囊癌亚群细胞并进行肿瘤干细胞特性分析,鉴定出胆囊癌干细胞样细胞。
方法：将新鲜的人胆囊癌组织标本切成小块后置入裸鼠皮下建立裸鼠荷瘤模型,为下一步研究提供胆囊癌标本材料；将胆囊癌组织和GBC-SD细胞制成单细胞悬液后,标上抗人CD24-FITC、CD44-APC和CD133-PE流式抗体,流式细胞术检测并分离出CD24+、CD44+和CD133+细胞,进一步研究各亚群的生物学特性；将具有肿瘤干细胞特性的亚群标志物联合检测、分选亚群细胞及研究生物学特性包括在无血清培养环境中克隆形成球、对化疗药吉西他滨和5-氟尿嘧啶的敏感性、在NOD/SCID鼠体内形成异种移植瘤能力及体内自我更新、分化潜能。
结果：新鲜的人胆囊癌组织标本植入裸鼠皮下3-6月后大部分裸鼠成功长出移植瘤；经流式分析CD24+、CD44+和CD133+细胞在原代胆囊癌和GBC-SD细胞中的比例分别为：20.83%-35.72%和55.73%,78.19%～93.36%和96.78%,1.93%-3.43%和61.28%；经生物学特性研究,CD24+和CD24-细胞在形成克隆球、移植瘤形成能力上没有明显差别,差异性没有统计学意义(P>0.05),而CD44+和CD133+细胞在形成克隆球、移植瘤形成能力上均明显强于相应的阴性亚群(P<0.05)；流式进一步检测观察到CD44+CD133+细胞在原代肿瘤和GBC-SD细胞中的比例为2.53%和60.62%,且CD44+CD133+细胞较CD44+CD133-细胞具有更强的形成克隆球、耐受化疗和移植瘤形成能力；经流式分析和免疫组化检测CD44+CD133+细胞形成的移植瘤细胞中,既有CD44+和CD133+细胞,也含有CD44-和CD133-细胞。
结论：人胆囊癌CD44+CD133+细胞具有肿瘤干细胞的特性,其中富集有肿瘤干细胞,CD44和CD133可用来分离胆囊干细胞。
第三部分CD133特异siRNA对胆囊癌细胞生物学行为的影响及核转录因子Gli在胆囊癌细胞中的表达
目的：通过运用RNAi干扰技术沉默胆囊癌细胞株GBC-SD的CD133的表达,研究其对GBC-SD细胞的生物学行为的影响,研究核转录因子Gli在胆囊癌细胞中的表达。方法：将CD133特异性siRNA转染胆囊癌细胞株GBC-SD,并运用Western blot和流式细胞术分析并优化转染的效率和效果；在无血清环境中检测其克隆球形成能力,运用MTT分析其体外增殖和对5-氟尿嘧啶的耐受性,以细胞划痕实验检测其细胞迁徙能力；运用实时定量RT-PCR检测核转录因子Gli1、Gli2、Gli3在胆囊癌不同亚群中的表达。
结果：特异性siRNA转染后,经Western blot和流式细胞术分析,胆囊癌细胞株GBC-SD的CD133表达明显下调；转染CD133特异性siRNA组细胞较空白对照组和转染非特异性siRNA组细胞,其细胞克隆球形成能力、细胞增殖能力及细胞迁徙能力均明显下降(P<0.05),而对5-氟尿嘧啶的敏感性无明显差异(P>0.05)；核转录因子Gli1在胆囊癌CD44+CD133+细胞中表达上调。
结论：CD133与胆囊癌干细胞的增殖和细胞迁徙相关,可作为胆囊癌的治疗靶点之一；Glil可能参与胆囊癌干细胞生物学特性的调控。
Part I Culture of floating tumor spheres from human gallbladder carcinoma in serum-free media and study of the Biological characteristics
Objective To culture and expand the floating tumor spheres from human gallbladder carcinoma cells in vitro and study the biological characteristics of the sphere forming cells, and to determine whether cancer stem cells were contained in the gallbladder carcinoma and filter the cell surface markers preliminary.
Methods Human primary gallbladder carcinoma specimens and gallbladder cancer cell line GBC-SD were made into single cancer cells and cultured to grow into tumor spheres in serum-free DMEM/F12 media which contained some cell factors. The tumor spheres were passaged and expanded. Then differentiation analysis was performed by culturing in media containing serum and stem markers including Oct-4 and Nestin were detected using real time RT-PCR. MTT method was used to measure proliferation capacity and sensitivity to chemotherapeutic drugs, and tumorigenicity was measured by transplanted into the nude mice. Then the expression of CD24, CD44 and CD133 in spheres forming cells and adherent cells was tested using flow cytometry.
Results Both primary human gallbladder cancer cells and GBC-SD cells were found to grow into spheres in the serum-free environment and the sphere forming cells could reform new spheres. The floating tumor spheres became adherent and cells migrated from the spheres, and the spheres became smaller and disappeared in the differentiating environment. The expression of Oct-4 and Nestin was down-regulated in the adherent cells. Compared with the adherent cells, sphere forming cells showed the higher proliferation capacity, chemoresistance to gemcitabine and 5-fluorouracil and higher tumorigenicity. CD133+cells showed a higher proportion in sphere forming cells than in adherent cells.
Conclusion Human gllbladder carcinoma cells contain cancer stem cellst and the cancer stem cells could be expanded to some extent in stem cells environment, and CD 133 protein may be the cell surface marker of the cancer stem cells.
Part II Isolation and identification of cancer stem cells in human gallbladder carcinoma
Objective To isolate and identify the cancer stem cells in gallbladder carcinoma using CD24,CD44 and CD133 proteins.
Methods Fresh tissue samples of human gallbladder carcinoma were cut into smaller pieces and were transplanted into nude mice to established tumor model. Gallbladder carcinoma tissue and gallbladder cancer cell line GBC-SD were made into single cells and labeled anti-human CD24-FITC, CD44-APC and CD133-PE antibody. The CD24+,CD44+ and CD133+subset cells were isolated and the biological characteristics was studied. Then the multiple-marker isolation was performed and the biological characteristics of subset cells including sphere formation, sensitivity to chemotherapeutic drugs, tumorigenicity, self-renewal and differentiation potential were studied.
Results A majority of mouse tumor models were successfully established.The proportion of CD24+,CD44+ and CD133+ subset cells were 20.83%～35.72% and 55.73%, 78.19%～93.36% and 96.78%,1.93%～3.43% and 61.28%, in primary gallbladder carcinoma and cell line GBC-SD respectively. For biological characteristics, CD24+ and CD24 cells showed no significant difference in spheroid formation capacity or in tumorigenicity (P>0.05).While CD44+ and CD133+ gallbladder carcinoma cells showed higher spheroid formation capacity and tumorigenicity than their crresponding negative subsets (P<0.05). The proportion of CD44+ CD133+ subset was 2.53% and 60.62% in primary gallbladder carcinoma and cell line GBC-SD respectively. Compared to CD44+ CD 133, CD44+ CD 133+ subset cells showed higher spheroid formation capacity, chemoresistance and tumorigenicity (P<0.05). Both flow cytometry and immunohistochemistry showed that the transplantable tumors derived from CD44+ CD133+ cells contained not only CD44+ CD133+ cells,but CD44-and CD133-subsets.
Conclusion CD44+CD133cells exhibited cancer stem cells characteristics in human gallbladder carcinoma and the cancer stem cells were enriched in this subset cells.
PartⅢInfluence of CD133-specific siRNA on the biological behaviors of gallbladder cancer cells and expression of transcription factor Glis in gallbladder cancer cells
Objective To study the role of CD133 in gallbladder cancer cells by using CD133-specific siRNA to silence CD 133 expresion in GBC-SD.And to study the expression of of transcription factor Glis in gallbladder cancer cells.
Methods CD133-specific siRNA was transfected into gallbladder cancer cell line GBC-SD, and Western blot and flow cytometry were used to analyze and optimize the efficiency and effectiveness of transfection. Then tumor sphere formation capacity was detected in serum-free environment, and proliferation capacity and sensitivity to 5-Fu were tested using MTT method. A cell wounding assay was performed to analyze cell migration. Expression of transcription factors Glil, Gli2, Gli3 in CD44+CD133+cell and others subsets was detected using real time RT-PCR.
Results Expression of CD 133 was down-regulated after specific siRNA transfection. Compared with control cells, those transfected specific siRNA showed a lower tumor sphere formation, proliferation and cell migration capacity (P<0.05), while chemoresistance showed no significant change(P>0.05). Nuclear transcription factor Glil expression was upregulated in CD44+CD133+gallbladder cancer cells.
Conclusion CD 133 was associated with proliferation and migration of CSCs in GBC and Glil may be involved in the regulation of biological characteristics of gallbladder cancer stem cells.
引文
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