背景:研究表明壳聚糖可间接促进成纤维细胞的增生和胶原的合成.利用壳聚糖和特定的成纤维细胞一起构建肌腱损伤修复的组织工程材料,以期在促进损伤修复的同时防止粘连.目的:观察水溶性壳聚糖对3T3TK成纤维细胞生长及表型的影响.设计:观察对照实验.单位:九江学院医学院.材料:3T3TK成纤维细胞由中国科学院细胞库提供.水溶性壳聚糖(规格:60目、30CPS,济南海得贝海洋生物工程有限公司),DMEM(低糖)(美国GIBCO公司),胎牛血清(杭州四季青生物工程研究所),青霉素、链霉素(美国Biosharp公司),胰蛋白酶(北京博大泰克生物基因技术有限责任公司).方法:实验于2004-11/2006-04在九江学院医学科研中心的系统生物学临床应用实验室(江西省高校重点实验室)完成.①常规培养3T3TK成纤维细胞至对数生长期,制成单细胞悬液,接种96孔板,共设5组,每组5孔,共25孔,每孔加细胞悬液200 μL,培养24 h后弃上清,前4组各孔分别加入含不同浓度壳聚糖的DMEM培养液200 μL,壳聚糖终浓度分别为1,0.1,0.01,0.001 g/L,第5组为阴性对照组,加不含壳聚糖的DMEM培养液,采用CellTilter-GloTM细胞活力测试盒检测不同浓度壳聚糖对成纤维细胞活力的影响.②常规培养细胞至对数生长期,制成单细胞悬液,接种24孔板,共设4组,前3组每孔分别加入含1,0.1,0.01 g/L壳聚糖的DMEM培养液1 mL,第4组为阴性对照组.采用羟脯氨酸、碱性磷酸酶试剂盒分别检测细胞培养上清液的胶原与碱性磷酸酶的含量.主要观察指标:①水溶性壳聚糖对体外培养成纤维细胞活力影响.②细胞培养上清液的胶原与碱性磷酸酶的含量.结果:①细胞活力检测结果:不同浓度壳聚糖组与阴性对照组相比,细胞活力差异均无统计学意义(P＞0.05).②细胞培养上清液的胶原与碱性磷酸酶的含量:1 g/L和0.1 g/L浓度壳聚糖组细胞培养上清液中的羟脯氨酸含量分别为(7.598±0.770),(8.366±0.308)mg/L,高于阴性对照组[(11.269±0.707)mg/L,P＜0.01];胶原含量分别为(56.703±5.748),(62.437±2.301)mg/L,低于阴性对照组[(84.099±5.280)mg/L,P＜0.01].,随壳聚糖浓度的上升,胶原含量下降,呈剂量依赖.与阴性对照组相比,各浓度壳聚糖组细胞培养上清液中碱性磷酸酶活性均无显著差异(P＞0.05).结论:水溶性壳聚糖并不明显抑制3T3TK成纤维细胞的活力,但能使其分泌胶原的量显著减少,提示壳聚糖具有作为肌腱修复的组织工程材料,并抑制肌腱修复中粘连形成的潜力.
揹景:研究錶明殼聚糖可間接促進成纖維細胞的增生和膠原的閤成.利用殼聚糖和特定的成纖維細胞一起構建肌腱損傷脩複的組織工程材料,以期在促進損傷脩複的同時防止粘連.目的:觀察水溶性殼聚糖對3T3TK成纖維細胞生長及錶型的影響.設計:觀察對照實驗.單位:九江學院醫學院.材料:3T3TK成纖維細胞由中國科學院細胞庫提供.水溶性殼聚糖(規格:60目、30CPS,濟南海得貝海洋生物工程有限公司),DMEM(低糖)(美國GIBCO公司),胎牛血清(杭州四季青生物工程研究所),青黴素、鏈黴素(美國Biosharp公司),胰蛋白酶(北京博大泰剋生物基因技術有限責任公司).方法:實驗于2004-11/2006-04在九江學院醫學科研中心的繫統生物學臨床應用實驗室(江西省高校重點實驗室)完成.①常規培養3T3TK成纖維細胞至對數生長期,製成單細胞懸液,接種96孔闆,共設5組,每組5孔,共25孔,每孔加細胞懸液200 μL,培養24 h後棄上清,前4組各孔分彆加入含不同濃度殼聚糖的DMEM培養液200 μL,殼聚糖終濃度分彆為1,0.1,0.01,0.001 g/L,第5組為陰性對照組,加不含殼聚糖的DMEM培養液,採用CellTilter-GloTM細胞活力測試盒檢測不同濃度殼聚糖對成纖維細胞活力的影響.②常規培養細胞至對數生長期,製成單細胞懸液,接種24孔闆,共設4組,前3組每孔分彆加入含1,0.1,0.01 g/L殼聚糖的DMEM培養液1 mL,第4組為陰性對照組.採用羥脯氨痠、堿性燐痠酶試劑盒分彆檢測細胞培養上清液的膠原與堿性燐痠酶的含量.主要觀察指標:①水溶性殼聚糖對體外培養成纖維細胞活力影響.②細胞培養上清液的膠原與堿性燐痠酶的含量.結果:①細胞活力檢測結果:不同濃度殼聚糖組與陰性對照組相比,細胞活力差異均無統計學意義(P＞0.05).②細胞培養上清液的膠原與堿性燐痠酶的含量:1 g/L和0.1 g/L濃度殼聚糖組細胞培養上清液中的羥脯氨痠含量分彆為(7.598±0.770),(8.366±0.308)mg/L,高于陰性對照組[(11.269±0.707)mg/L,P＜0.01];膠原含量分彆為(56.703±5.748),(62.437±2.301)mg/L,低于陰性對照組[(84.099±5.280)mg/L,P＜0.01].,隨殼聚糖濃度的上升,膠原含量下降,呈劑量依賴.與陰性對照組相比,各濃度殼聚糖組細胞培養上清液中堿性燐痠酶活性均無顯著差異(P＞0.05).結論:水溶性殼聚糖併不明顯抑製3T3TK成纖維細胞的活力,但能使其分泌膠原的量顯著減少,提示殼聚糖具有作為肌腱脩複的組織工程材料,併抑製肌腱脩複中粘連形成的潛力.
배경:연구표명각취당가간접촉진성섬유세포적증생화효원적합성.이용각취당화특정적성섬유세포일기구건기건손상수복적조직공정재료,이기재촉진손상수복적동시방지점련.목적:관찰수용성각취당대3T3TK성섬유세포생장급표형적영향.설계:관찰대조실험.단위:구강학원의학원.재료:3T3TK성섬유세포유중국과학원세포고제공.수용성각취당(규격:60목、30CPS,제남해득패해양생물공정유한공사),DMEM(저당)(미국GIBCO공사),태우혈청(항주사계청생물공정연구소),청매소、련매소(미국Biosharp공사),이단백매(북경박대태극생물기인기술유한책임공사).방법:실험우2004-11/2006-04재구강학원의학과연중심적계통생물학림상응용실험실(강서성고교중점실험실)완성.①상규배양3T3TK성섬유세포지대수생장기,제성단세포현액,접충96공판,공설5조,매조5공,공25공,매공가세포현액200 μL,배양24 h후기상청,전4조각공분별가입함불동농도각취당적DMEM배양액200 μL,각취당종농도분별위1,0.1,0.01,0.001 g/L,제5조위음성대조조,가불함각취당적DMEM배양액,채용CellTilter-GloTM세포활력측시합검측불동농도각취당대성섬유세포활력적영향.②상규배양세포지대수생장기,제성단세포현액,접충24공판,공설4조,전3조매공분별가입함1,0.1,0.01 g/L각취당적DMEM배양액1 mL,제4조위음성대조조.채용간포안산、감성린산매시제합분별검측세포배양상청액적효원여감성린산매적함량.주요관찰지표:①수용성각취당대체외배양성섬유세포활력영향.②세포배양상청액적효원여감성린산매적함량.결과:①세포활력검측결과:불동농도각취당조여음성대조조상비,세포활력차이균무통계학의의(P＞0.05).②세포배양상청액적효원여감성린산매적함량:1 g/L화0.1 g/L농도각취당조세포배양상청액중적간포안산함량분별위(7.598±0.770),(8.366±0.308)mg/L,고우음성대조조[(11.269±0.707)mg/L,P＜0.01];효원함량분별위(56.703±5.748),(62.437±2.301)mg/L,저우음성대조조[(84.099±5.280)mg/L,P＜0.01].,수각취당농도적상승,효원함량하강,정제량의뢰.여음성대조조상비,각농도각취당조세포배양상청액중감성린산매활성균무현저차이(P＞0.05).결론:수용성각취당병불명현억제3T3TK성섬유세포적활력,단능사기분비효원적량현저감소,제시각취당구유작위기건수복적조직공정재료,병억제기건수복중점련형성적잠력.
BACKGROUND:Studies have demonstrated that chitosan can indirectly promote the proliferation of fibroblast and the synthesis of collagen, Using chitosan and specific fibroblasts together to construct tissue-engineering materials for tendon repair may be an adequate way to promote healing and prevent adhesion during the healing process.OBJECTIVE: To observe the effect of water soluble chitosan (WSC) on the growth and genotype of 3T3TK fibroblasts.DESIGN: Controlled experiment based on observation.SETTING: Jiujiang University Medical College.MATERIALS: 3T3TK fibroblasts were provided by Cell Bank of Chinese Academy of Sciences. WSC (specification: 60 mesh, 30CPS, Jinan Haidebei Marine Bioengineering Co.,Ltd),DMEM (low sugar) (Gibco Company, USA), fetal bovine serum (FBS, Sijiqing Biological Engineering Institute, Hangzhou), penicillin, streptomycin (Biosharp Company, USA),trypsin (BioEev-Tech Scientific & Technical Co.,Ltd, Beijing).METHODS: This experiment was carried out in the Laboratory for Clinical Application of Biology, Center for Medial Scientific Research, Jiujiang University between November 2004 and April 2006. ①Cells in the log phase were digested with 2.5 g/L trypsin to produce single cell suspension and inoculated into a 96-well culture plate at a density of 6 000 cells /200 μL medium. Five groups were prepared, 5 holes per group. 200 μL cell suspension was added into each well.After 24 hours of cultivation, supernatant was discarded, 200 μL DMEM with 1, 0.1, 0.01 and 0.000 1 g/L chitosan was added respectively in the first four groups. The remaining group was taken as the negative control group, in which 200 μL DMEM medium without chitosan was added. The cell suspension was routinely cultured for 72 hours. The cell viability was measured by CellTilter- GloTM Luminescent Cell Viability Assay according to the manufacturer's protocol. Cells in the log phase were split and inoculated into 24-well culture plates. Four groups were prepared (5 holes per group). 1 mL DMEM medium supplemented with 1, 0.1, 0.01 g/L chitosan was added into the first 3 groups respectively, and the 4th group was set as control group. Hydroxyproline and alkaline phosphatase(ALP) kits were used for detecting the contents of collagen and ALP in the supernatant of fibroblasts.MAIN OUTCOME MEASURES : ①Effect of WSC on viability of fibroblasts cultured in vitro.②Contents of collagen and ALP in the cell culture supernatant of fibroblasts.RESULTS:①Detection results of viability of fibroblasts: There were no significant differences in viability of fibroblasts between each chitosan group and control group (P ＞ 0.05).②Contents of collagen and ALP in the cell culture supernatant of fibroblasts: Hydroxyproline content in the cell culture supernatant of fibroblasts of 1 g/L and 0.1 g/L groups was (7.598±0.770) and (8.366±0.308)mg/L, respectively, which was higher than that of control group [(11.269±0.707)mg/L,P ＜ 0.01]; Collagen content in the 1 g/L and 0.1 g/L groups was(56.703±5.748) and(62.437±2.301)mg/L, respectively, which was lower than that of control group [(84.099±5.280)mg/L,P ＜ 0.01]. With the concentration of chitosan increasing, the collagen content was decreased in a dose-dependent manner. There were no significant differences in ALP activity in the cell culture supernatant between each chitosan group and control group (P ＞0.05).CONCLUSION: WSC does not obviously inhibit the viability of 3T3TK fibroblasts, but can markedly reduce the content of secreted collagen. It is indicated that chitosan can be used as tissue engineering material for tendon repair, and inhibit adhesion formation during tendon repair.
