中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2009年
50期
9892-9897
,共6页
曾润铭%杜世新%吴杰%罗绍伟%刘东昕%王虎
曾潤銘%杜世新%吳傑%囉紹偉%劉東昕%王虎
증윤명%두세신%오걸%라소위%류동흔%왕호
昆明山海棠%类风湿关节炎%滑膜细胞%增殖%凋亡
昆明山海棠%類風濕關節炎%滑膜細胞%增殖%凋亡
곤명산해당%류풍습관절염%활막세포%증식%조망
背景:昆明山海棠应用于类风湿关节炎治疗已被证实有确切疗效,但对于其作用机制目前尚不明确.目的:验证昆明山海棠对类风湿关节炎滑膜巨噬样细胞和成纤维样细胞体外增殖活性及对其凋亡影响,分析其剂量效应关系.设计、时间及地点:细胞学体外分组对照实验,于2003-08/2007-06在汕头大学医学院第一附属医院中心实验室和南方医科大学细胞生物实验室完成.材料:类风湿关节炎6例及正常滑膜组织3例,来源于汕头大学医学院第一附属医院骨科和南方医院脊柱骨病科患者术中获得的标本.昆明山海棠制备水提取液,含生药0.667 g/mL..方法:收集获得的滑膜组织,通过消化法获得原代类风湿关节炎及正常滑膜细胞,第2-4代细胞用免疫磁珠法筛选滑膜CD68~+细胞和CD68~-细胞,在接种72 h后加入实验各组分别在培养液中加入最终体积分数分别为5,10,20 mL/L的昆明山海棠药液处理24 h和48 h.同时设立不加药物的阴性对照及不加细胞空白对照.主要观察指标:相差倒置显微镜、扫描电镜观察细胞形态,用MTT法测定细胞增殖情况,流式细胞仪检测和TUNEL.法检测细胞凋亡.结果:分选后滑膜CD68~-细胞为成纤维样细胞,滑膜CD68~+细胞为滑膜巨噬样细胞.施予干预因素后,各组有部分细胞体积缩小,胞膜皱缩,微绒毛和伪足减少或消失,部分细胞可见团块脱落形成凋亡小体.当昆明山海棠体积分数为2%时,各组细胞均有不同程度抑制,显示昆明山海棠有细胞毒性,对类风湿关节炎滑膜CD68~-、CD68~+细胞的抑制效应和诱导细胞效应强于同种正常滑膜细胞(P<0.01).在药物体积分数为1%时,对类风湿关节炎滑膜细胞有明显抑制效应(P<0.01).昆明山海棠对诱导类风湿关节炎滑膜细胞凋亡作用有明显时间依赖性和剂量依赖性,且凋亡率与相同条件下的抑制率表现出较好的直线相关关系(r=0.497,P<0.01).结论:昆明山海棠对于类风湿关节炎滑膜巨噬样细胞和滑膜成纤维样细胞有诱导凋亡和明显抑制异常增殖作用.在一定浓度下,昆明山海棠对于正常滑膜细胞毒性较小且对类风湿关节炎滑膜细胞的有较明显抑制异常增殖和诱导细胞凋亡的效应.
揹景:昆明山海棠應用于類風濕關節炎治療已被證實有確切療效,但對于其作用機製目前尚不明確.目的:驗證昆明山海棠對類風濕關節炎滑膜巨噬樣細胞和成纖維樣細胞體外增殖活性及對其凋亡影響,分析其劑量效應關繫.設計、時間及地點:細胞學體外分組對照實驗,于2003-08/2007-06在汕頭大學醫學院第一附屬醫院中心實驗室和南方醫科大學細胞生物實驗室完成.材料:類風濕關節炎6例及正常滑膜組織3例,來源于汕頭大學醫學院第一附屬醫院骨科和南方醫院脊柱骨病科患者術中穫得的標本.昆明山海棠製備水提取液,含生藥0.667 g/mL..方法:收集穫得的滑膜組織,通過消化法穫得原代類風濕關節炎及正常滑膜細胞,第2-4代細胞用免疫磁珠法篩選滑膜CD68~+細胞和CD68~-細胞,在接種72 h後加入實驗各組分彆在培養液中加入最終體積分數分彆為5,10,20 mL/L的昆明山海棠藥液處理24 h和48 h.同時設立不加藥物的陰性對照及不加細胞空白對照.主要觀察指標:相差倒置顯微鏡、掃描電鏡觀察細胞形態,用MTT法測定細胞增殖情況,流式細胞儀檢測和TUNEL.法檢測細胞凋亡.結果:分選後滑膜CD68~-細胞為成纖維樣細胞,滑膜CD68~+細胞為滑膜巨噬樣細胞.施予榦預因素後,各組有部分細胞體積縮小,胞膜皺縮,微絨毛和偽足減少或消失,部分細胞可見糰塊脫落形成凋亡小體.噹昆明山海棠體積分數為2%時,各組細胞均有不同程度抑製,顯示昆明山海棠有細胞毒性,對類風濕關節炎滑膜CD68~-、CD68~+細胞的抑製效應和誘導細胞效應彊于同種正常滑膜細胞(P<0.01).在藥物體積分數為1%時,對類風濕關節炎滑膜細胞有明顯抑製效應(P<0.01).昆明山海棠對誘導類風濕關節炎滑膜細胞凋亡作用有明顯時間依賴性和劑量依賴性,且凋亡率與相同條件下的抑製率錶現齣較好的直線相關關繫(r=0.497,P<0.01).結論:昆明山海棠對于類風濕關節炎滑膜巨噬樣細胞和滑膜成纖維樣細胞有誘導凋亡和明顯抑製異常增殖作用.在一定濃度下,昆明山海棠對于正常滑膜細胞毒性較小且對類風濕關節炎滑膜細胞的有較明顯抑製異常增殖和誘導細胞凋亡的效應.
배경:곤명산해당응용우류풍습관절염치료이피증실유학절료효,단대우기작용궤제목전상불명학.목적:험증곤명산해당대류풍습관절염활막거서양세포화성섬유양세포체외증식활성급대기조망영향,분석기제량효응관계.설계、시간급지점:세포학체외분조대조실험,우2003-08/2007-06재산두대학의학원제일부속의원중심실험실화남방의과대학세포생물실험실완성.재료:류풍습관절염6례급정상활막조직3례,래원우산두대학의학원제일부속의원골과화남방의원척주골병과환자술중획득적표본.곤명산해당제비수제취액,함생약0.667 g/mL..방법:수집획득적활막조직,통과소화법획득원대류풍습관절염급정상활막세포,제2-4대세포용면역자주법사선활막CD68~+세포화CD68~-세포,재접충72 h후가입실험각조분별재배양액중가입최종체적분수분별위5,10,20 mL/L적곤명산해당약액처리24 h화48 h.동시설립불가약물적음성대조급불가세포공백대조.주요관찰지표:상차도치현미경、소묘전경관찰세포형태,용MTT법측정세포증식정황,류식세포의검측화TUNEL.법검측세포조망.결과:분선후활막CD68~-세포위성섬유양세포,활막CD68~+세포위활막거서양세포.시여간예인소후,각조유부분세포체적축소,포막추축,미융모화위족감소혹소실,부분세포가견단괴탈락형성조망소체.당곤명산해당체적분수위2%시,각조세포균유불동정도억제,현시곤명산해당유세포독성,대류풍습관절염활막CD68~-、CD68~+세포적억제효응화유도세포효응강우동충정상활막세포(P<0.01).재약물체적분수위1%시,대류풍습관절염활막세포유명현억제효응(P<0.01).곤명산해당대유도류풍습관절염활막세포조망작용유명현시간의뢰성화제량의뢰성,차조망솔여상동조건하적억제솔표현출교호적직선상관관계(r=0.497,P<0.01).결론:곤명산해당대우류풍습관절염활막거서양세포화활막성섬유양세포유유도조망화명현억제이상증식작용.재일정농도하,곤명산해당대우정상활막세포독성교소차대류풍습관절염활막세포적유교명현억제이상증식화유도세포조망적효응.
BACKGROUND:Tripterygium hypoglaucum(Levl.)Hutch (THH) has been accurately confirmed a good clinical therapeutic effect for rheumatoid arthritis(RA).However.functionaI mechanism of THH remains unclearly.OBJECTIVE:To investigate the effect of THH on proliferation and apoptosis of macrophage-like synoviocytes and fibroblast-like synoviocytes from patients with RA and to explore its dose-effect relation.DESIGN.TIME AND SETTING:In vitro cytology grouping controlled observation was performed in the Center Laboratory of the First Clinical College of Shantou University and the Laboratory for Cellular Biology of Southern MedicaI University from August 2003 to June 2007.MATERIALS:Synovium was obtained from 6 patients with RA and 3 normaI synovial samples.All samples sourced from patients admitted in Department of Orthopaedics.the First Clinical College of Shantou University and in Spinal and Joint Surgery of Nanfang Hospital.The water extracts of THH contained 0.667 g/mL crude drug.METHODS:Synovial cells were isolated by digesting synovial tissue with collagenase.CD68+and CD68-synoviocytes were sorted from synovial cells by Dynabeads(magnetic celI sorting)from the 2~(nd)-4~(th) passages of synoviocyes.After 72 hours incubation,all groups of isolated synoviocytes were cultured in culture medium containing the flnaI concentrations of 0 mL/L,5 mL/L,1 0 mL/L and 20 mL/L of THH for 24 and 48 hours,respectively,and the common culture medium was sewed as the control. MAIN OUTCOME MEASURES:The morphology of synoviocytes were observed by inverted phase contrast microscope and studied by scanning electron microscopy(SEM).In vitro cell growth was assessed by the MTT assay,and synoviocytes apoptosis was evaluated using temlinaI deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL)assay and a flow cytometry.RESULTS:CD68~+ synoviocytes were appeared macrophage-like and CD68~- synoviocytes were exhibited fibroblast-like.After incubated with THH,some synoviocytes presented the volume of cells deflated,progressively destruction of cell membrane,microvillus or pseudopodium tended to be decreased even disappeared,and apoptosis bodies appeared.When incubated with 2% THH,proliferation inhibiUon and inducing apoptosis could be observed;it showed that THH had the cytotoxic effect on synoviocytes.The effect of proliferation inhibition and inducing apoptosis on CD68-and CD68+synoviocytes from RA patients was more significantly than that from normal arthritis (P<0.01).When incubated with 1%THH.proliferation inhibition and inducing apoptosis on synoviocytes from RA patients also could be found(P<0.01),but not found on normal synoviocytes(P>0.05).Compared with the negative controI group.proliferation inhibition was no significant difference On all groups with the low dose (≤5 mL/L) of THH(P>0.05).Furthermore,the effect of proliferation inhibition and inducing apoptosis on synoviocytes decreased in a dose-dependent and time-dependent manner.The inhibition rate of synoviocytes also could be found positively correlated with apoptosis rate aftat treatment of THH(r=0.497,P<0.01).CONCLUSlON:THH have cytotoxic effect on rheumatoid synoviocytes and normal synoviocytes.it can inhibit macrophage-like synoviocytes and fibroblast-like synoviocytes from RA on proliferation and induce them to apoptosis.In the some contraction of THH.proliferation Inhibition and inducing apoptosis can be occur on synoviocytes from RA but not on normal synoviocytes.