间充质干细胞%癌,肝细胞%细胞增殖%细胞周期%培养基,条件性
間充質榦細胞%癌,肝細胞%細胞增殖%細胞週期%培養基,條件性
간충질간세포%암,간세포%세포증식%세포주기%배양기,조건성
Mesenchymal stem cells%Carcinoma,hepatocellular%Cell proliferation%Cell cycle%Culture media,conditioned
目的 探讨人脐带源间充质干细胞(HUC-MSC)条件培养液(CM)对人肝癌HepG2细胞生长的影响.方法 选择2011年2月至2014年1月于中国人民解放军第105医院妇产科分娩的35例足月妊娠剖宫产健康胎儿脐带为研究对象,取其用于分离、培养并扩增HUC-MSC.传代扩增至第3代HUC-MSC融合达80％时,更换培养液,继续培养24 h后收集上清液,即为HUC-MSC-CM备用.将对数生长期的人肝癌HepG2细胞按照随机数字表法随机分成相等的3份,分别于含50％,20％及不含HUC-MSC-CM的培养中培养,并分别纳入高含量组、低含量组和空白对照组.采用倒置显微镜观察人肝癌HepG2细胞生长状态,采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法和细胞划痕愈合实验分别检测3组人肝癌HepG2细胞增殖和迁移情况,并采用流式细胞术(FCM)法检测各组细胞的细胞周期变化情况.本研究遵循的程序符合中国人民解放军第105医院人体试验委员会所制定的伦理学标准,得到该委员会批准,脐带获取前,均征得孕妇知情同意,并与之签署临床研究知情同意书.结果 ①所分离的原代HUC-MSC培养至第3代后,细胞形态开始比较均一.②MTT法检测结果显示,人肝癌HepG2细胞培养24,48和72 h时,3组相对吸光度(A)值比较,差异均有统计学意义(F=21.330,6.223,9.345;P=0.004,0.032,0.027),且在这3个时间点,高含量组和低含量组相对A值均显著高于空白对照组,差异均有统计学意义(P＜0.05),高含量组相对A值亦显著高于低含量组,差异亦均有统计学意义(P＜0.05).③划痕愈合实验结果显示,3组人肝癌HepG2细胞过河时间比较,差异有统计学意义(F=12.860,P=0.025),且高含量组人肝癌HepG2细胞过河时间短于低含量组和空白对照组,差异均有统计学意义(P＜0.05),低浓度组人肝癌HePG2细胞过河时间亦短于对照组,差异亦有统计学意义(P＜0.05).④FCM法检测人肝癌HepG2细胞周期结果显示,3组G0/G1期和S期人肝癌HepG2细胞比例比较,差异均有统计学意义(F=30.600,30.590;P＜0.05).且与空白对照组相比,高含量组和低含量组人肝癌HepG2细胞G0/G1期细胞比例显著降低,S期细胞比例显著增加,差异均有统计学意义(P＜0.01);且高含量组G0/G1期细胞比例比低含量组下降更显著,而S期细胞比例显著增加,差异均有统计学意义(P＜0.05).结论 HUC-MSC-CM可促进入肝癌HepG2细胞增殖和迁移,并可促进人肝癌HepG2细胞周期G1/S转换.
目的 探討人臍帶源間充質榦細胞(HUC-MSC)條件培養液(CM)對人肝癌HepG2細胞生長的影響.方法 選擇2011年2月至2014年1月于中國人民解放軍第105醫院婦產科分娩的35例足月妊娠剖宮產健康胎兒臍帶為研究對象,取其用于分離、培養併擴增HUC-MSC.傳代擴增至第3代HUC-MSC融閤達80％時,更換培養液,繼續培養24 h後收集上清液,即為HUC-MSC-CM備用.將對數生長期的人肝癌HepG2細胞按照隨機數字錶法隨機分成相等的3份,分彆于含50％,20％及不含HUC-MSC-CM的培養中培養,併分彆納入高含量組、低含量組和空白對照組.採用倒置顯微鏡觀察人肝癌HepG2細胞生長狀態,採用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴鹽(MTT)法和細胞劃痕愈閤實驗分彆檢測3組人肝癌HepG2細胞增殖和遷移情況,併採用流式細胞術(FCM)法檢測各組細胞的細胞週期變化情況.本研究遵循的程序符閤中國人民解放軍第105醫院人體試驗委員會所製定的倫理學標準,得到該委員會批準,臍帶穫取前,均徵得孕婦知情同意,併與之籤署臨床研究知情同意書.結果 ①所分離的原代HUC-MSC培養至第3代後,細胞形態開始比較均一.②MTT法檢測結果顯示,人肝癌HepG2細胞培養24,48和72 h時,3組相對吸光度(A)值比較,差異均有統計學意義(F=21.330,6.223,9.345;P=0.004,0.032,0.027),且在這3箇時間點,高含量組和低含量組相對A值均顯著高于空白對照組,差異均有統計學意義(P＜0.05),高含量組相對A值亦顯著高于低含量組,差異亦均有統計學意義(P＜0.05).③劃痕愈閤實驗結果顯示,3組人肝癌HepG2細胞過河時間比較,差異有統計學意義(F=12.860,P=0.025),且高含量組人肝癌HepG2細胞過河時間短于低含量組和空白對照組,差異均有統計學意義(P＜0.05),低濃度組人肝癌HePG2細胞過河時間亦短于對照組,差異亦有統計學意義(P＜0.05).④FCM法檢測人肝癌HepG2細胞週期結果顯示,3組G0/G1期和S期人肝癌HepG2細胞比例比較,差異均有統計學意義(F=30.600,30.590;P＜0.05).且與空白對照組相比,高含量組和低含量組人肝癌HepG2細胞G0/G1期細胞比例顯著降低,S期細胞比例顯著增加,差異均有統計學意義(P＜0.01);且高含量組G0/G1期細胞比例比低含量組下降更顯著,而S期細胞比例顯著增加,差異均有統計學意義(P＜0.05).結論 HUC-MSC-CM可促進入肝癌HepG2細胞增殖和遷移,併可促進人肝癌HepG2細胞週期G1/S轉換.
목적 탐토인제대원간충질간세포(HUC-MSC)조건배양액(CM)대인간암HepG2세포생장적영향.방법 선택2011년2월지2014년1월우중국인민해방군제105의원부산과분면적35례족월임신부궁산건강태인제대위연구대상,취기용우분리、배양병확증HUC-MSC.전대확증지제3대HUC-MSC융합체80％시,경환배양액,계속배양24 h후수집상청액,즉위HUC-MSC-CM비용.장대수생장기적인간암HepG2세포안조수궤수자표법수궤분성상등적3빈,분별우함50％,20％급불함HUC-MSC-CM적배양중배양,병분별납입고함량조、저함량조화공백대조조.채용도치현미경관찰인간암HepG2세포생장상태,채용3-(4,5-이갑기새서-2)-2,5-이분기사담서추염(MTT)법화세포화흔유합실험분별검측3조인간암HepG2세포증식화천이정황,병채용류식세포술(FCM)법검측각조세포적세포주기변화정황.본연구준순적정서부합중국인민해방군제105의원인체시험위원회소제정적윤리학표준,득도해위원회비준,제대획취전,균정득잉부지정동의,병여지첨서림상연구지정동의서.결과 ①소분리적원대HUC-MSC배양지제3대후,세포형태개시비교균일.②MTT법검측결과현시,인간암HepG2세포배양24,48화72 h시,3조상대흡광도(A)치비교,차이균유통계학의의(F=21.330,6.223,9.345;P=0.004,0.032,0.027),차재저3개시간점,고함량조화저함량조상대A치균현저고우공백대조조,차이균유통계학의의(P＜0.05),고함량조상대A치역현저고우저함량조,차이역균유통계학의의(P＜0.05).③화흔유합실험결과현시,3조인간암HepG2세포과하시간비교,차이유통계학의의(F=12.860,P=0.025),차고함량조인간암HepG2세포과하시간단우저함량조화공백대조조,차이균유통계학의의(P＜0.05),저농도조인간암HePG2세포과하시간역단우대조조,차이역유통계학의의(P＜0.05).④FCM법검측인간암HepG2세포주기결과현시,3조G0/G1기화S기인간암HepG2세포비례비교,차이균유통계학의의(F=30.600,30.590;P＜0.05).차여공백대조조상비,고함량조화저함량조인간암HepG2세포G0/G1기세포비례현저강저,S기세포비례현저증가,차이균유통계학의의(P＜0.01);차고함량조G0/G1기세포비례비저함량조하강경현저,이S기세포비례현저증가,차이균유통계학의의(P＜0.05).결론 HUC-MSC-CM가촉진입간암HepG2세포증식화천이,병가촉진인간암HepG2세포주기G1/S전환.
Objective To investigate the effect of human umbilical cord-derived mesenchymal stem cells (HUC-MSC) condition medium (CM) on cell growth of hepatocellular carcinoma HepG2 cell.Methods From February 2011 to January 2014,a total of 35 cases of full-term pregnancy healthy fetuses' umbilical cords were chose into this study.All the fetuses were delivered through uterine-incision in the 105th Hospital of People's Liberation Army.Mesenchymal stem cells were derived from human umbilical cord and expanded in vitro.When the confluence of the third generation reached 80％,the medium was changed to fresh medium.After 24-hour incubation,the supernatant HUC-MSC-CM was collected.Then the HepG2 cells in logarithmic growth phase were divided into 3 equal groups randomly by random number table method.They were cultured with 5 ％ fetal calf serum high glucose (HG)-DMEM fresh medium alone as the control group,and the experimental group were cultured with 25％ HUC-MSC-CM together with 5％ fetal calf serum HG-DMEM (low concentration group) and 50％ MSC-CM together with 5％ fetal calf serum HG-DMEM (high concentration group),respectively.HepG2 morphology was observed by inverted microscope.The abilities of proliferation were detected by 3-(4,5-dimethyl-thiazol-2)-2,5-diphenyl tetrazolium bromide (MTT) method,and the migration of HepG2 cells in each group were detected by wound healing assay.The cell cycle distribution was analyzed by flow cytometry (FCM).The study protocol was approved by the Ethical Reviews Board of Investigation in Human Being of the 105th Hospital of People's Liberation Army.Informed consent was obtained from all participants.Results ① The morphology of isolated primary HUC-MSC was uniform when cultured into the third generation.②MTT assay showed that when the HepG2 cells were cultured 24,48 and 72 h,the differences of relative absorbance (A) values in the three groups were statistically significant (F=21.330,6.223,9.345;P=0.004,0.032,0.027).And the relative A values of HepG2 cells in two experimental groups significantly increased than that in control group at these three time points,and the differences were statistically significant (P ＜ 0.05);the relative A values of HepG2 cells in high concentrations group was also significantly higher than in low concentration group,and the difference was also statistically significant (P＜ 0.05).③)Wound healing assay showed that the difference of healing time of HepG2 cell in three groups was statistically significant (F=12.860,P=0.025),and the healing time in high concentration group was shorter than that in the low concentration group and control group,the differences were statistically significant (P＜0.05);the healing time in the low concentration group was shorter than that in the control group,the difference was statistically significant (P ＜ 0.05).④ FCM assay showed that there were significant differences among the percentage of cells in G0/G1 phase and S phase in three groups (F =30.600,30.590;P＜0.05).And compared with control group,the percentage of HepG2 cells in G0/G1 phase of high concentration group and low concentration group were significantly decrease,and the percentage of cells in S phase were higher,all the differences were statistically significant (P＜0.01).The percentage of cells in G0/G1 phase of the high concentration group decreased more significantly than that in low concentration group,while the percentage of cells in S phase increased more significantly,the differences were statistically significant (P ＜ 0.05).Conclusions HUC-MSC-CM could promote the proliferation and migration of HepG2 cells,and it can accelerate cell cycle G1/S phase transition.
