【摘要】：目的：研究證實(shí),骨髓基質(zhì)細(xì)胞(bone mesenchymal stem cells, BMSCs)在表皮生長(zhǎng)因子(epidermal growth factor,EGF)和堿性成纖維生長(zhǎng)因子(basic fibroblast growth factor,bFGF)誘導(dǎo)下可向神經(jīng)樣細(xì)胞分化,具有促進(jìn)脊髓損傷(Spinal cord injury, SCI)修復(fù)的作用,但由于EGF、bFGF進(jìn)入體內(nèi)后迅速擴(kuò)散、變性或酶解,無(wú)法保持其生物活性,難以達(dá)到體內(nèi)持續(xù)誘導(dǎo)骨髓基質(zhì)細(xì)胞向神經(jīng)樣細(xì)胞分化的作用。運(yùn)用殼聚糖緩釋微球負(fù)載細(xì)胞因子能夠達(dá)到抵御人體代謝的作用。本實(shí)驗(yàn)的目的是制備殼聚糖緩釋微球負(fù)載EGF和bFGF,在體外培養(yǎng)條件下應(yīng)用于BMSCs中,觀察：1、EGF-bFGF-殼聚糖緩釋微球?qū)MSCs活性、增殖能力的影響；2、誘導(dǎo)BMSCs分化為神經(jīng)樣細(xì)胞的情況,為進(jìn)一步聯(lián)合BMSCs、 EGF-bFGF-殼聚糖緩釋微球進(jìn)行體內(nèi)移植治療SCI提供理論依據(jù)。方法：1.骨髓基質(zhì)細(xì)胞的體外培養(yǎng)及鑒定：采用貼壁篩選法對(duì)取自成年S-D大鼠股骨和脛骨的BMSCs進(jìn)行分離培養(yǎng),并用細(xì)胞免疫熒光方法鑒定BMSCs的干細(xì)胞標(biāo)記物CD44、CD90,骨髓造血干細(xì)胞標(biāo)記物CD45。2. EGF-bFGF-殼聚糖緩釋微球的制備及檢測(cè)采用乳化交聯(lián)法,制備EGF-bFGF-殼聚糖緩釋微球。采用elisa方法進(jìn)行EGF和bFGF的檢測(cè),計(jì)算載藥率、包裝率及微球的緩釋率。流式細(xì)胞術(shù)檢測(cè)空白殼聚糖緩釋微球?qū)MSCs的細(xì)胞毒性。3. EGF-bFGF-殼聚糖緩釋微球誘導(dǎo)BMSCs向神經(jīng)元樣細(xì)胞分化的觀察：準(zhǔn)備如下分組：a、對(duì)照組,BMSCs+DMEM;b、空白殼聚糖緩釋微球組,BMSCs+DMEM+空白殼聚糖緩釋微球；c、EGF/bFGF直接給藥組,BMSCs+DMEM+bFGF(終濃度lOng/ml)+EGF(終濃度20ng/ml);d、EGF-bFGF-殼聚糖緩釋微球組,BMSCs+DMEM+EGF-bFGF-殼聚糖緩釋微球細(xì)胞(使之終釋放濃度與C組相同)；光鏡下觀察BMSCs細(xì)胞學(xué)形態(tài)的變化,MTT比色法檢測(cè)BMSCs的生長(zhǎng)增殖情況,進(jìn)一步利用免疫熒光檢測(cè)神經(jīng)前體細(xì)胞標(biāo)記物Nestin、神經(jīng)元特異性烯醇化酶NSE和星形膠質(zhì)細(xì)胞標(biāo)記物GFAP染色情況。結(jié)果：1、原代培養(yǎng)的骨髓基質(zhì)細(xì)胞10-14d時(shí)細(xì)胞融合成片,細(xì)胞形態(tài)多呈長(zhǎng)梭形。經(jīng)4-5次傳代,細(xì)胞純度較高,主要以呈細(xì)長(zhǎng)梭形的BMSCs為主。第5代體外培養(yǎng)BMSCs用細(xì)胞免疫熒光方法進(jìn)行鑒定,觀察到細(xì)胞呈CD44和CD90陽(yáng)性表達(dá),CD45呈陰性表達(dá)。2、乳化交聯(lián)法進(jìn)行緩釋微球的制備,經(jīng)ELISA法檢測(cè)得知：殼聚糖終濃度為2mg/ml時(shí),平均載藥量最高；而緩釋微球的包裝率隨著TPP濃度的升高逐漸升高,當(dāng)其濃度為0.45mg/ml時(shí),包封率達(dá)到峰值。EGF和bFGF在PH7.4和PH5.8的溶劑中,24h內(nèi)均出現(xiàn)了一個(gè)約40-60%的突釋過(guò)程,隨后開(kāi)始緩慢釋放。3、采用流式細(xì)胞結(jié)合Annexin V-FITC/PI雙染法檢測(cè)細(xì)胞的凋亡情況,正常對(duì)照組和殼聚糖緩釋微球處理組24h后細(xì)胞凋亡率無(wú)顯著性差異(P0.05)。MTT法檢測(cè)BMSCs的生長(zhǎng)增殖情況,四組BMSCs細(xì)胞存活率的比較結(jié)果：與對(duì)照組相比,空白殼聚糖緩釋微球組的細(xì)胞增殖率沒(méi)有統(tǒng)計(jì)學(xué)差異(P0.05),EGF/bFGF組和EGF-bFGF-殼聚糖緩釋微球組其細(xì)胞存活率在相同時(shí)間點(diǎn)均較對(duì)照組和空白殼聚糖緩釋微球組顯著升高(P0.05)。Annexin V-FITC/PI雙染法檢測(cè)結(jié)果表明空白殼聚糖緩釋微球無(wú)生物學(xué)毒性。MTT法檢測(cè)結(jié)果表明空白殼聚糖緩釋微球作為載體對(duì)BMSCs的存活無(wú)顯著影響,EGF/bFGF組和EGF-bFGF-殼聚糖緩釋微球組可促進(jìn)BMSCs的生長(zhǎng)增殖。4、四組BMSCs誘導(dǎo)培養(yǎng)5天后,空白殼聚糖緩釋微球組和對(duì)照組均未發(fā)生變化,仍然維持BMSCs的典型形態(tài)。EGF/bFGF直接給藥組和EGF-bFGF-殼聚糖緩釋微球組部分細(xì)胞呈現(xiàn)神經(jīng)細(xì)胞樣的改變。高內(nèi)涵免疫熒光檢測(cè)：EGF/bFGF組與EGF-bFGF-殼聚糖緩釋微球組：Nestin、NSE、GFAP熒光染色均有陽(yáng)性表達(dá)。共聚焦免疫熒光鑒定：EGF/bFGF組：(36.51±2.53)%的細(xì)胞表達(dá)神經(jīng)前體細(xì)胞標(biāo)記物Nestin, (17.66±3.44)%的細(xì)胞表達(dá)神經(jīng)元細(xì)胞標(biāo)志物NSE,(27.39±3.03)%的細(xì)胞表達(dá)星形膠質(zhì)細(xì)胞標(biāo)記物GFAP; EGF-bFGF-殼聚糖緩釋微球組：(30.87±3.53)%的細(xì)胞表達(dá)神經(jīng)前體細(xì)胞標(biāo)記物Nestin,(10.57-2.24)%的細(xì)胞表達(dá)神經(jīng)元細(xì)胞標(biāo)志物NSE,(21.31±4.69)%的細(xì)胞表達(dá)星形膠質(zhì)細(xì)胞標(biāo)記物GFAP,結(jié)論：1、采用貼壁篩選法對(duì)大鼠BMSCs進(jìn)行培養(yǎng)是一種簡(jiǎn)單易行的方法,可迅速獲得大量純度較高的BMSCs.2、乳化交聯(lián)法制備EGF-bFGF-殼聚糖緩釋微球,工藝簡(jiǎn)單,包封率高,載藥量大,在中性PH值條件下有良好的緩釋性能,對(duì)BMSCs無(wú)生物學(xué)毒性,可以促進(jìn)BMSCs的生長(zhǎng)增殖。3. EGF-bFGF-殼聚糖緩釋微球能誘導(dǎo)骨髓基質(zhì)細(xì)胞分化為神經(jīng)樣細(xì)胞。4.EGF-bFGF-殼聚糖緩釋微球誘導(dǎo)BMSCs分化為神經(jīng)樣細(xì)胞的效率較直接EGF/bFGF低,可能與EGF-bFGF-殼聚糖緩釋微球釋放細(xì)胞因子的初始濃度低有關(guān)。EGF-bFGF-殼聚糖緩釋微球誘導(dǎo)BMSCs分化的最佳濃度有待進(jìn)一步檢測(cè)。
[Abstract]:Objective : To study the effects of bone marrow stromal cells ( MSCs ) on the differentiation of nerve - like cells under the induction of epidermal growth factor ( EGF ) and basic fibroblast growth factor ( bFGF ) .
Methods : 1 . In vitro culture and identification of bone marrow stromal cells : 1 . In vitro culture and identification of bone marrow stromal cells : 1 . In vitro culture and identification of bone marrow stromal cells : The stem cell marker CD44 , CD90 and bone marrow hematopoietic stem cell marker CD45.2 were identified by cell immunofluorescence method . The preparation and detection of EGF - bFGF - chitosan sustained - release microspheres by emulsion crosslinking method were used to prepare the EGF - bFGF - chitosan sustained - release microspheres . The detection of EGF and bFGF was carried out by the enzyme - linked immunosorbent assay ( ELISA ) , the drug loading rate , the packing ratio and the slow release rate of microspheres were calculated . The cytotoxicity of the blank chitosan sustained - release microspheres was detected by flow cytometry . The effects of EGF - bFGF - chitosan sustained - release microspheres on neuron - like cell differentiation were observed .
c , EGF / bFGF direct drug administration group , bone marrow + DMEM + bFGF ( final concentration lOng / ml ) + EGF ( final concentration 20ng / ml ) ; d , EGF - bFGF - chitosan sustained - release microsphere group , bone marrow + DMEM + EGF - bFGF - chitosan sustained - release microsphere cells ( the final release concentration is the same as group C ) ;
The growth and proliferation of bone marrow stromal cells ( Nestin , NSE ) and astrocytes ( GFAP ) were detected by MTT colorimetric assay .
There was no significant difference between EGF / bFGF group and EGF - bFGF - chitosan sustained - release microspheres ( P0.05 ) . EGF - bFGF - chitosan sustained - release microspheres can induce differentiation of bone marrow stromal cells into neural - like cells .
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R651.2

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