成纤维细胞%细胞,培养的%细胞衰老%β半乳糖苷酶类
成纖維細胞%細胞,培養的%細胞衰老%β半乳糖苷酶類
성섬유세포%세포,배양적%세포쇠로%β반유당감매류
背景:二倍体成纤维细胞常用于研究细胞水平的衰老,β-半乳糖苷酶是鉴别衰老细胞的标志酶,本实验拟观察正常人成纤维细胞在体外培养过程中β-半乳糖苷酶的变化情况.目的:建立一个稳定的二倍体成纤维细胞体外培养体系,了解正常人成纤维细胞老化过程中β-半乳糖苷酶的变化规律.设计:自身对照实验.单位:潍坊医学院整形外科研究所、潍坊医学院附属医院普外科.材料:实验于2000-09/2002-09在潍坊医学院整形外科研究所完成.实验样本来源于在潍坊医学院附属医院普外科行包皮环切术的6～8岁正常男性儿童(n=20)切除的健康包皮组织.方法:①成纤维细胞培养:取包皮组织,分离真皮和表皮,真皮部分用含100 mL/L胎牛血清DMEM培养液终止胰蛋白酶作用,再用200 U/mLⅠ型胶原酶37℃条件下消化30 min,收集细胞悬液,接种于培养皿中,细胞培养达80%汇合时进行传代培养.②细胞鉴定:用相差显微镜观察细胞形态变化及生长增殖情况、透射电镜及抗vimentin免疫细胞化学染色.③细胞生物学行为的观察对比:取生长状态良好的细胞,按群体倍增水平分为两组,第一组代表年轻细胞,采用10代龄;第二组代表老化细胞,采用65代龄.进行消化传代,分别按2×104,4×104分别接种于24孔培养板内.每隔24 h消化计数,每次每组计数3孔,计算均值.共记数8 d,以培养时间为横轴,细胞数为纵轴,绘制生长曲线并计算细胞的群体倍增时间.④β-半乳糖苷酶组化检测及阳性细胞的半定量分析:成纤维细胞每5 n代龄(共13代)作细胞爬片,漂洗,加入SA-β-gal染色液,37℃孵育48 h.在显微镜下观察计数,蓝染细胞为阳性细胞,每次随机挑选视野计数500个细胞,得到阳性细胞百分率.主要观察指标:①正常人成纤维细胞生物学行为的改变.②正常人成纤维细胞老化过程中β-半乳糖苷酶的表达.结果:①正常人成纤维细胞生物学行为的改变:细胞生长曲线显示,老化细胞生长速度较年轻细胞明显减慢.年轻细胞最大增殖数约为47.3×105,对数生长期在3.0～6.0 d,细胞倍增时间为2.18 d.老化细胞最大增殖数各为8.5×104,对数生长期分别在4.0～7.5 d,细胞群体倍增时间依次为3.86d.②正常人成纤维细胞老化过程中β-半乳糖苷酶的表达:与年轻细胞组(阳性细胞率=4%)相比,衰老细胞组中阳性细胞率(60%)大为增强.直线相关分析结果显示,β-半乳糖苷酶染色的阳性率和细胞代龄之间呈显著正相关(r=0.92,P＜0.01).结论:在正常人成纤维细胞从年轻向老化发展的过程中,细胞群体倍增时间呈增高的趋势;与年轻细胞相比较(占4%),β-半乳糖苷酶的表达在老化细胞中显著增强(占60%),且这种增强与细胞衰老表型的出现和细胞增殖能力的丧失相平行,可反应细胞的老化程度.本实验为建立成纤维细胞衰老模型提供了实验依据,同时也为老年学及肿瘤生物学甚至组织工程学发展提供科学依据.
揹景:二倍體成纖維細胞常用于研究細胞水平的衰老,β-半乳糖苷酶是鑒彆衰老細胞的標誌酶,本實驗擬觀察正常人成纖維細胞在體外培養過程中β-半乳糖苷酶的變化情況.目的:建立一箇穩定的二倍體成纖維細胞體外培養體繫,瞭解正常人成纖維細胞老化過程中β-半乳糖苷酶的變化規律.設計:自身對照實驗.單位:濰坊醫學院整形外科研究所、濰坊醫學院附屬醫院普外科.材料:實驗于2000-09/2002-09在濰坊醫學院整形外科研究所完成.實驗樣本來源于在濰坊醫學院附屬醫院普外科行包皮環切術的6～8歲正常男性兒童(n=20)切除的健康包皮組織.方法:①成纖維細胞培養:取包皮組織,分離真皮和錶皮,真皮部分用含100 mL/L胎牛血清DMEM培養液終止胰蛋白酶作用,再用200 U/mLⅠ型膠原酶37℃條件下消化30 min,收集細胞懸液,接種于培養皿中,細胞培養達80%彙閤時進行傳代培養.②細胞鑒定:用相差顯微鏡觀察細胞形態變化及生長增殖情況、透射電鏡及抗vimentin免疫細胞化學染色.③細胞生物學行為的觀察對比:取生長狀態良好的細胞,按群體倍增水平分為兩組,第一組代錶年輕細胞,採用10代齡;第二組代錶老化細胞,採用65代齡.進行消化傳代,分彆按2×104,4×104分彆接種于24孔培養闆內.每隔24 h消化計數,每次每組計數3孔,計算均值.共記數8 d,以培養時間為橫軸,細胞數為縱軸,繪製生長麯線併計算細胞的群體倍增時間.④β-半乳糖苷酶組化檢測及暘性細胞的半定量分析:成纖維細胞每5 n代齡(共13代)作細胞爬片,漂洗,加入SA-β-gal染色液,37℃孵育48 h.在顯微鏡下觀察計數,藍染細胞為暘性細胞,每次隨機挑選視野計數500箇細胞,得到暘性細胞百分率.主要觀察指標:①正常人成纖維細胞生物學行為的改變.②正常人成纖維細胞老化過程中β-半乳糖苷酶的錶達.結果:①正常人成纖維細胞生物學行為的改變:細胞生長麯線顯示,老化細胞生長速度較年輕細胞明顯減慢.年輕細胞最大增殖數約為47.3×105,對數生長期在3.0～6.0 d,細胞倍增時間為2.18 d.老化細胞最大增殖數各為8.5×104,對數生長期分彆在4.0～7.5 d,細胞群體倍增時間依次為3.86d.②正常人成纖維細胞老化過程中β-半乳糖苷酶的錶達:與年輕細胞組(暘性細胞率=4%)相比,衰老細胞組中暘性細胞率(60%)大為增彊.直線相關分析結果顯示,β-半乳糖苷酶染色的暘性率和細胞代齡之間呈顯著正相關(r=0.92,P＜0.01).結論:在正常人成纖維細胞從年輕嚮老化髮展的過程中,細胞群體倍增時間呈增高的趨勢;與年輕細胞相比較(佔4%),β-半乳糖苷酶的錶達在老化細胞中顯著增彊(佔60%),且這種增彊與細胞衰老錶型的齣現和細胞增殖能力的喪失相平行,可反應細胞的老化程度.本實驗為建立成纖維細胞衰老模型提供瞭實驗依據,同時也為老年學及腫瘤生物學甚至組織工程學髮展提供科學依據.
배경:이배체성섬유세포상용우연구세포수평적쇠로,β-반유당감매시감별쇠로세포적표지매,본실험의관찰정상인성섬유세포재체외배양과정중β-반유당감매적변화정황.목적:건립일개은정적이배체성섬유세포체외배양체계,료해정상인성섬유세포노화과정중β-반유당감매적변화규률.설계:자신대조실험.단위:유방의학원정형외과연구소、유방의학원부속의원보외과.재료:실험우2000-09/2002-09재유방의학원정형외과연구소완성.실험양본래원우재유방의학원부속의원보외과행포피배절술적6～8세정상남성인동(n=20)절제적건강포피조직.방법:①성섬유세포배양:취포피조직,분리진피화표피,진피부분용함100 mL/L태우혈청DMEM배양액종지이단백매작용,재용200 U/mLⅠ형효원매37℃조건하소화30 min,수집세포현액,접충우배양명중,세포배양체80%회합시진행전대배양.②세포감정:용상차현미경관찰세포형태변화급생장증식정황、투사전경급항vimentin면역세포화학염색.③세포생물학행위적관찰대비:취생장상태량호적세포,안군체배증수평분위량조,제일조대표년경세포,채용10대령;제이조대표노화세포,채용65대령.진행소화전대,분별안2×104,4×104분별접충우24공배양판내.매격24 h소화계수,매차매조계수3공,계산균치.공기수8 d,이배양시간위횡축,세포수위종축,회제생장곡선병계산세포적군체배증시간.④β-반유당감매조화검측급양성세포적반정량분석:성섬유세포매5 n대령(공13대)작세포파편,표세,가입SA-β-gal염색액,37℃부육48 h.재현미경하관찰계수,람염세포위양성세포,매차수궤도선시야계수500개세포,득도양성세포백분솔.주요관찰지표:①정상인성섬유세포생물학행위적개변.②정상인성섬유세포노화과정중β-반유당감매적표체.결과:①정상인성섬유세포생물학행위적개변:세포생장곡선현시,노화세포생장속도교년경세포명현감만.년경세포최대증식수약위47.3×105,대수생장기재3.0～6.0 d,세포배증시간위2.18 d.노화세포최대증식수각위8.5×104,대수생장기분별재4.0～7.5 d,세포군체배증시간의차위3.86d.②정상인성섬유세포노화과정중β-반유당감매적표체:여년경세포조(양성세포솔=4%)상비,쇠로세포조중양성세포솔(60%)대위증강.직선상관분석결과현시,β-반유당감매염색적양성솔화세포대령지간정현저정상관(r=0.92,P＜0.01).결론:재정상인성섬유세포종년경향노화발전적과정중,세포군체배증시간정증고적추세;여년경세포상비교(점4%),β-반유당감매적표체재노화세포중현저증강(점60%),차저충증강여세포쇠로표형적출현화세포증식능력적상실상평행,가반응세포적노화정도.본실험위건립성섬유세포쇠로모형제공료실험의거,동시야위노년학급종류생물학심지조직공정학발전제공과학의거.
BACKGROUND :Diploid fibroblasts are commonly used in study of senescence at the cellular level, and β-galactosidase is the marker enzyme of cell aging. This experiment is aimed to observe the changes of β-galactosidase in fibroblasts during in vitro culture.OBJECTIVE: To establish a stablein vitro culture system for diploid fibroblasts and observe the changes of β-galactosidase in normal fibroblasts during the aging process.DESIGN: Self-control experiment.SETTING: Institute of Plastic Surgery, Weifang Medical College; Department of General Surgery, Affiliated Hospital of Weifang Medical College.MATERIALS: This experiment was carried ont at the Institute of Plastic Surgery, Weifang Medical College between September 2000 and September 2002. Samples were obtained from normal prepuce tissues (n=20) of boys aged 6-8 years old who received peritomy at the Department of General Surgery, Affiliated Hospital of Weifang Medical College. Informed consent was obtained.and then dermis and epidermis were separated, the latter was digested with trypsin. For the dermis, DMEM containing 100 mL/L FBS was used to terminate the reaction before digested with 200 U/mL collagenase I at 37℃ for 30 minutes, cells were collected and inoculated in the culture dish; when cell congregation reached 80%, they were digested and reinocmorphological changes and growth of cells were observed under the inverted phase contrast microscope; the transmission electron microscope parison of cytobiological behavior: The well-growing cells were divided into two groups according to the level of clone multiplication. The first group represented young cells, using 10 generations of age; the second group represented aged cells, using 65 generations of age. The cells were digested and inoculated in 24-well culture dishes by 2×104 and 4×104,respectively. Cells were digested and counted once every 24 hours, 3wells in each group were counted each time for calculating the average,altogether for 8 days. Taking the culture days as the abscissa axis and cell number as the ordinate axis, growth curve was drawn and population dase and semi-quantitative analysis of positive cells: Fibroblasts were used for slide attaching by every 5n generation age , rinsed and stained with SA- β-gal at 37 ℃ for 48 hours. Blue colored positive cells were counted under the microscope to calculate the percentage of positive cells in randomly selected 500 cells within one field of vision, which repre- sented cell aging rate.man fibroblasts during the aging process.cording to the cell growth curve, aging cells grew obviously more slowly than the younger ones. The maximum multiplication value of young cells was 47.3×105, with logarithm growth period varying within 3.0-6.0 days and cell multiplication time of 2.18 days, as compared to the corresponding galactosidase in normal human fibroblasts during the aging process: The positive cell rate in aging cell group greatly increased (60%) as compared to that of young cells (4%). The linear correlation analysis revealed that β-galactosidase positive cells had remarkably positive correlation with cell generation age (r=0.92, P ＜ 0.01).CONCLUSION: Cell clone multiplication time was found on an increasing tendency with normal fibroblasts developing from young to senile. In contrast with that of young cells (4%), the expression of β-galactosidase in aging cells (60%) was remarkably enhanced, which paralleled with the appearance of cell senile phenotype and cell proliferation, and can be used to reflect the degree of cell aging. This experiment provides experimental basis for the establishment of fibroblast aging model; meanwhile it also provides scientific evidence for the development of tumor biology and organic engineering.
