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
28期
5444-5448
,共5页
陈欣戬%闫福华%钟泉%赵欣%江一平
陳訢戩%閆福華%鐘泉%趙訢%江一平
진흔전%염복화%종천%조흔%강일평
碱性成纤维细胞生长因子%基因治疗%成纤维细胞%组织工程
堿性成纖維細胞生長因子%基因治療%成纖維細胞%組織工程
감성성섬유세포생장인자%기인치료%성섬유세포%조직공정
背景:研究发现,局部应用外源性碱性成纤维细胞生长因子可明显促进体外培养牙龈成纤维细胞的增殖,口内局部应用可加速牙龈组织伤口的愈合.目的:观察碱性成纤维细胞生长因子基因转染对Beagle犬牙龈成纤维细胞生物学特性的影响.设计、时间、地点:观察对比体外细胞学实验,于2008-04/09在福建医科大学细胞与发育工程中心及解放军南京军区福州总医院比较医学科完成.材料:Beagle犬4只,12月龄,体质量10～13 kg,雄性;含有人全长碱性成纤维细胞生长因子cDNA的pIRES2-EGFP-bFGF质粒为白行构建.方法:切取Beagle犬左上颌第2,3,4前磨牙的游离龈.无菌磷酸盐缓冲液冲洗4遍,将组织块剪碎后用2.5 g/L的胰酶37℃消化2 h,离心后弃上清,加入含体积分数为10%胎生血清的DMEM,接种于6孔板并覆盖玻片,置于37℃、体积分数为5%CO<,2>的培养箱培养,取对数生长期细胞消化传代.利用脂质体介导法将pIRES2-EGFP-bFGF质粒转染Beagle犬牙龈成纤维细胞,以空质粒转染组及未转染组作为对照.主要观察指标:MTT法检测牙龈成纤维细胞增殖状况;AO/EB双染色检测牙龈成纤维细胞凋亡;化学比色法检测牙龈成纤维细胞碱性磷酸酶活性.结果:3组细胞均在转染后第3天开始进入对数生长期.MTT检测结果显示,转染后随着时间改变,与空质粒转染组及未转染组相比,实验组牙龈成纤维细胞的增殖能力明显增强(P<0.05),而空质粒转染组及未转染组差异无显著性意义(P>0.05).AO/EB双染色结果显示,实验组牙龈成纤维细胞凋亡率低于空质粒转染组及未转染组(P<0.05).转染碱性成纤维细胞生长因子基因后,牙龈成纤维细胞碱性磷酸酶活性未见明显改变,与未转染细胞差异无显著性意义.结论:碱性成纤维细胞生长因子基因转染可以加速牙龈成纤维细胞的增殖,抑制其凋亡,无促使牙龈成纤维细胞骨向分化的作用.
揹景:研究髮現,跼部應用外源性堿性成纖維細胞生長因子可明顯促進體外培養牙齦成纖維細胞的增殖,口內跼部應用可加速牙齦組織傷口的愈閤.目的:觀察堿性成纖維細胞生長因子基因轉染對Beagle犬牙齦成纖維細胞生物學特性的影響.設計、時間、地點:觀察對比體外細胞學實驗,于2008-04/09在福建醫科大學細胞與髮育工程中心及解放軍南京軍區福州總醫院比較醫學科完成.材料:Beagle犬4隻,12月齡,體質量10～13 kg,雄性;含有人全長堿性成纖維細胞生長因子cDNA的pIRES2-EGFP-bFGF質粒為白行構建.方法:切取Beagle犬左上頜第2,3,4前磨牙的遊離齦.無菌燐痠鹽緩遲液遲洗4遍,將組織塊剪碎後用2.5 g/L的胰酶37℃消化2 h,離心後棄上清,加入含體積分數為10%胎生血清的DMEM,接種于6孔闆併覆蓋玻片,置于37℃、體積分數為5%CO<,2>的培養箱培養,取對數生長期細胞消化傳代.利用脂質體介導法將pIRES2-EGFP-bFGF質粒轉染Beagle犬牙齦成纖維細胞,以空質粒轉染組及未轉染組作為對照.主要觀察指標:MTT法檢測牙齦成纖維細胞增殖狀況;AO/EB雙染色檢測牙齦成纖維細胞凋亡;化學比色法檢測牙齦成纖維細胞堿性燐痠酶活性.結果:3組細胞均在轉染後第3天開始進入對數生長期.MTT檢測結果顯示,轉染後隨著時間改變,與空質粒轉染組及未轉染組相比,實驗組牙齦成纖維細胞的增殖能力明顯增彊(P<0.05),而空質粒轉染組及未轉染組差異無顯著性意義(P>0.05).AO/EB雙染色結果顯示,實驗組牙齦成纖維細胞凋亡率低于空質粒轉染組及未轉染組(P<0.05).轉染堿性成纖維細胞生長因子基因後,牙齦成纖維細胞堿性燐痠酶活性未見明顯改變,與未轉染細胞差異無顯著性意義.結論:堿性成纖維細胞生長因子基因轉染可以加速牙齦成纖維細胞的增殖,抑製其凋亡,無促使牙齦成纖維細胞骨嚮分化的作用.
배경:연구발현,국부응용외원성감성성섬유세포생장인자가명현촉진체외배양아간성섬유세포적증식,구내국부응용가가속아간조직상구적유합.목적:관찰감성성섬유세포생장인자기인전염대Beagle견아간성섬유세포생물학특성적영향.설계、시간、지점:관찰대비체외세포학실험,우2008-04/09재복건의과대학세포여발육공정중심급해방군남경군구복주총의원비교의학과완성.재료:Beagle견4지,12월령,체질량10～13 kg,웅성;함유인전장감성성섬유세포생장인자cDNA적pIRES2-EGFP-bFGF질립위백행구건.방법:절취Beagle견좌상합제2,3,4전마아적유리간.무균린산염완충액충세4편,장조직괴전쇄후용2.5 g/L적이매37℃소화2 h,리심후기상청,가입함체적분수위10%태생혈청적DMEM,접충우6공판병복개파편,치우37℃、체적분수위5%CO<,2>적배양상배양,취대수생장기세포소화전대.이용지질체개도법장pIRES2-EGFP-bFGF질립전염Beagle견아간성섬유세포,이공질립전염조급미전염조작위대조.주요관찰지표:MTT법검측아간성섬유세포증식상황;AO/EB쌍염색검측아간성섬유세포조망;화학비색법검측아간성섬유세포감성린산매활성.결과:3조세포균재전염후제3천개시진입대수생장기.MTT검측결과현시,전염후수착시간개변,여공질립전염조급미전염조상비,실험조아간성섬유세포적증식능력명현증강(P<0.05),이공질립전염조급미전염조차이무현저성의의(P>0.05).AO/EB쌍염색결과현시,실험조아간성섬유세포조망솔저우공질립전염조급미전염조(P<0.05).전염감성성섬유세포생장인자기인후,아간성섬유세포감성린산매활성미견명현개변,여미전염세포차이무현저성의의.결론:감성성섬유세포생장인자기인전염가이가속아간성섬유세포적증식,억제기조망,무촉사아간성섬유세포골향분화적작용.
BACKGROUND: Studies have demonstrated that exogenous basic fibroblast growth factor (bFGF) has intensive effects to promote proliferation of gingival fibroblasts (GFs) cultured in vitro and the heeling of gingival wounds. OBJECTIVE: To investigate the effects of bFGF gene transfection on the biological performance of Beagle canine GFs. DESIGN, TIME AND SETTING: An observation and comparison in vitro experiment regarding cells was accomplished in Centre of Cell Biology and Development of Fujian Medical University and Department of Comparative Medicine in Fuzhou General Hospital of Nanjing Military Area Command of Chinese PLA from April to September of 2008. MATERIALS: Four beagle dogs, male, 12 months old, weighing 10-13 kg were used in this experiment, plRES2-EGFP-bFGF plasmid containing full-length human bFGF gene cDNA was constructed and conserved by our institution. METHODS: Free gingiva of the 2nd, 3rd and 4th premolars were excised from left upper jaw of Beagle dogs, dnsed with aseptic phosphate buffer four times, then cut into pieces and digested with 2.5 g/L pancreetin for 2 hours at 37 ℃. After the cantrifugation and supernatant removal, DMEM containing 10% fetal bovine serum was added to incubate on 6-well plate with coverlips in 5% CO2 incubator at 37 ℃. Logadthrnically growing cells were digested and passaged. GFs were transfected with pIRES2-EGFP-bFGF plasmid using liposome mediated method, while vacant plasmid transfection and un-transfection group served as controls.MAIN OUTCOME MEASURES: Proliferation and apoptosis feature of the GFs were evaluated by M'rE and AOEB, respectively. The activity of alkaline phosphatase was assayed by chemical coledmetry. RESULTS: All of three groups cells entered log phrase on three days after transfection. MTT results showed that the proliferation of GFs transfected with bFGF was greater than cells transfected with vacant vector and untransfected cells (P < 0.05). AO/EB dyeing showed the apoptosis rate of GFs transfected with bFGF was reduced compared with other two groups (P < 0.05). After bGFG gene transfection, the ALP activity remained unchanged and there was no significant difference compared with untransfected cells.CONCLUSION: The transfection of bFGF gene to GFs can promote the proliferation of GFs and depress the apoptosis. No promotion is present with regard to the GFs differentiation.
