發(fā)布時(shí)間：2018-01-02 06:21

  本文關(guān)鍵詞：不同濃度堿性成纖維細(xì)胞生長(zhǎng)因子對(duì)體外培養(yǎng)肌腱細(xì)胞增殖的影響　出處：《寧波大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腱細(xì)胞 細(xì)胞增殖 堿性成纖維細(xì)胞生長(zhǎng)因子 細(xì)胞培養(yǎng)

【摘要】：目的隨著對(duì)肌腱愈合機(jī)制研究的不斷深入及分子生物學(xué)等的不斷發(fā)展，發(fā)現(xiàn)多種生長(zhǎng)因子及其受體在肌腱愈合過(guò)程中起著關(guān)鍵的調(diào)控作用，其中堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor，bFGF)在臨床中促創(chuàng)面愈合已得到廣泛應(yīng)用，且臨床療效已得到證實(shí)，使其已成為研究促肌腱愈合的外源性生長(zhǎng)因子的熱點(diǎn)。本實(shí)驗(yàn)的目的在于探討不同濃度的bFGF對(duì)體外培養(yǎng)兔肌腱細(xì)胞增殖的影響，選擇出促進(jìn)肌腱細(xì)胞增殖的最佳濃度，并比較未凍存與凍存后復(fù)蘇細(xì)胞體外培養(yǎng)增殖差異，從而為肌腱損傷的修復(fù)和組織工程肌腱種子細(xì)胞的培養(yǎng)提供重要的基礎(chǔ)參數(shù)。 方法1、無(wú)菌條件下切取新西蘭乳兔的雙下肢趾屈肌腱，在顯微鏡下剝離肌腱外膜，采用Henderson分步酶消化法分離出肌腱細(xì)胞，并用含20%胎牛血清的F-12培養(yǎng)液進(jìn)行培養(yǎng)、傳代并凍存第2代腱細(xì)胞；2、倒置顯微鏡下觀察細(xì)胞形態(tài)變化及生長(zhǎng)情況，，并對(duì)所得肌腱細(xì)胞行免疫細(xì)胞化學(xué)染色法測(cè)定細(xì)胞合成膠原類型；3、在體外培養(yǎng)第2代兔肌腱細(xì)胞的培養(yǎng)液中分別加入不同濃度（0.5ng/ml、1ng/ml、2ng/ml、5ng/ml、10ng/ml、20ng/ml、30ng/ml、40ng/ml和50ng/ml）的bFGF，繼續(xù)培養(yǎng)48h，MTT法檢測(cè)不同濃度bFGF組光密度(optical density,OD)值，選出促肌腱細(xì)胞增殖的最佳bFGF濃度；4、復(fù)蘇凍存1月后的第2代肌腱細(xì)胞，使用最佳濃度的bFGF繼續(xù)體外培養(yǎng)，測(cè)定OD值，比較未凍存細(xì)胞與凍存后復(fù)蘇細(xì)胞增殖的差異；5、試驗(yàn)數(shù)據(jù)用SPSS18.0統(tǒng)計(jì)軟件包進(jìn)行統(tǒng)計(jì)學(xué)分析，以P0.05作為判斷差異有統(tǒng)計(jì)學(xué)意義的標(biāo)準(zhǔn)。 結(jié)果1、通過(guò)不同的酶消化分離可獲得較為純正的肌腱細(xì)胞，體外培養(yǎng)肌腱細(xì)胞可表現(xiàn)出良好的細(xì)胞增殖能力和傳代能力；2、用鼠抗兔膠原I、Ⅲ抗體染色，DAB顯色試劑盒顯色，肌腱細(xì)胞表現(xiàn)為陽(yáng)性反應(yīng)，證明所獲細(xì)胞為肌腱細(xì)胞；3、相比于對(duì)照組OD均值：5ng/ml、10ng/ml、20ng/ml、30ng/ml、40ng/ml和50ng/mlbFGF組差異有顯著性(P 0.05)；隨著bFGF濃度的增加，各組OD值先逐漸增大(5～20ng/mL),達(dá)到最高值（20ng/mL）后,又逐漸降低(20～50ng/mL)；4、應(yīng)用最佳促肌腱細(xì)胞增殖bFGF濃度后，凍存組和凍存后復(fù)蘇組之間細(xì)胞增殖無(wú)統(tǒng)計(jì)學(xué)差異（P0.05）。 結(jié)論1、肌腱細(xì)胞能夠在體外分離、擴(kuò)增和傳代，為研究肌腱愈合及構(gòu)建組織工程化人工肌腱所需種子細(xì)胞的獲取提供了可靠的試驗(yàn)基礎(chǔ)；2、bFGF有明顯促肌腱細(xì)胞增殖的作用，且與濃度有一定相關(guān)性，即促肌腱細(xì)胞增殖的起始bFGF濃度為5ng/ml，而20ng/ml時(shí)可達(dá)到促肌腱細(xì)胞增殖的最佳濃度；3、肌腱細(xì)胞凍存復(fù)蘇后并不會(huì)影響其體外培養(yǎng)增殖。
[Abstract]:Objective with the development of the mechanism of tendon healing and molecular biology, found that several growth factors and their receptors in tendon healing process play a key role, including basic fibroblast growth factor (basic fibroblast, growth factor, bFGF) in the clinical wound healing promotion has been widely used, and the clinical the effect has been confirmed, it has become a study of promoting tendon healing of exogenous growth factors focus. The purpose of this experiment is to study the effect of different concentration of bFGF on proliferation of rabbit tendon cells in vitro culture influence, choose the best concentration to stimulate the proliferation of tendon cells, and compare the unfrozen and frozen thawed cells in vitro the proliferation of differences, so as to provide essential parameters for training and repair of seed cells for tendon tissue engineering tendon injury.
Methods 1 harvested neonatal New Zealand rabbit leg flexor tendon, tendon membrane stripping under the microscope, isolated tendon cells by Henderson step enzyme digestion method and cultured with F-12 containing 20% fetal bovine serum culture, subculture and cryopreservation of the second generation of tendon cells; 2, cell morphology was observed change and growth under the microscope, and the collagen synthesis was determined by cell type the tendon cells by immunocytochemical staining; 3, cultured with different concentration were cultured in second generations of rabbit tendon cells in vitro (0.5ng/ml, 1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml and 50ng/ml) bFGF and continue to culture 48h, MTT method was used to detect the different concentration of bFGF groups (optical density, OD optical density) value, select the best concentration of bFGF to promote the proliferation of tendon cells; 4, resuscitated in January after the second generation of muscle tendon cells, with the optimal concentration of bFGF To determine OD value culture, in vitro, comparison of unfrozen between cells and frozen thawed cell proliferation; 5, the test data were analyzed using SPSS18.0 statistical software package, using P0.05 as the judgment standard. The difference was statistically significant
The 1, can obtain more pure tendon cells by enzyme digestion of different separation, tendon cells can show good cell proliferation and passage culture in vitro; 2, using mouse anti rabbit antibody staining of collagen I, III, DAB coloration kit, tendon cells showed positive reaction, that the cells were tendon cells; 3, compared to the control group mean OD 10ng/ml, 20ng/ml, 5ng/ml, 30ng/ml, there was significant difference between 40ng/ml and 50ng/mlbFGF group (P 0.05); with the increase of bFGF concentration, the group OD value gradually increased (5 ~ 20ng/mL), reached the highest value (20ng/mL), and gradually reduce (20 ~ 50ng/mL); 4, application of the best to promote proliferation of tendon cells bFGF concentration after cryopreservation group and frozen thawed group had no significant difference between cell proliferation (P0.05).
Conclusion 1, tendon cells in vitro isolation, amplification and passages, and provide the experimental basis for the study of tendon healing and reliable construction for tissue engineered tendon for seed cells; 2, bFGF can obviously promote the proliferation of tendon cells, and the concentration has certain correlation, i.e. the initial concentration of bFGF tendon cell proliferation of 5ng/ml, and 20ng/ml can reach the optimal concentration of promoting the proliferation of tendon cells; 3, tendon cells after cryopreservation does not affect its proliferation in vitro.

【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329.2

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