本文選題：骨髓基質(zhì)干細(xì)胞 + 骨形態(tài)發(fā)生蛋白-2��； 參考：《山東大學(xué)》2010年碩士論文

【摘要】： 目的： 骨形態(tài)發(fā)生蛋白(Bone Morphogenetic Protein 2,BMP2)是目前已知的具有最強(qiáng)促進(jìn)異位成骨能力細(xì)胞因子,血管內(nèi)皮生長(zhǎng)因子(Vascular endothelial growth factor, VEGF)能顯著促進(jìn)血管再生有利于骨缺損的愈合。BMP2和VEGF兩種蛋白的分泌存在相互促進(jìn)的正反饋?zhàn)饔谩９侨睋p的愈合,尤其是在愈合過程的初期,是在低氧環(huán)境下進(jìn)行的。骨髓基質(zhì)干細(xì)胞(Bone Marrow Stromal Cells,BMSC)具有向骨形成細(xì)胞和血管形成細(xì)胞等多種細(xì)胞分化的潛能。本課題研究模擬低氧環(huán)境下同時(shí)轉(zhuǎn)染hBMP2和VEGF基因的BMSC在成骨方面的相關(guān)特性。 方法： 1.大鼠骨髓基質(zhì)干細(xì)胞(BMSC)的分離、培養(yǎng)利用Percoll分離液(相對(duì)密度1.077),用密度梯度離心法從大鼠骨髓中分離骨髓基質(zhì)干細(xì)胞,進(jìn)行體外培養(yǎng)及擴(kuò)增。 2.通過形態(tài)學(xué)觀察和生長(zhǎng)曲線的測(cè)定及Elisa,觀察BMSC轉(zhuǎn)染hBMP-2的腺病毒載體(Ad-hBMP-2)和VEGF的腺病毒載體(Ad-VEGF)后生物學(xué)行為的變化和目的基因的表達(dá)。 BMSC轉(zhuǎn)染綠色熒光蛋白(Green Fluorescent Protein,GFP)的腺病毒載體(Ad-GFP)后,使用熒光顯微鏡來測(cè)定腺病毒的轉(zhuǎn)染效率,并觀測(cè)轉(zhuǎn)染Ad-GFP的細(xì)胞傳代后綠色熒光蛋白的表達(dá)情況。 3.MTT法檢測(cè)低氧控制系統(tǒng)下,轉(zhuǎn)染Ad-hBMP-2和VEGF的BMSC細(xì)胞增值及活力 取第二代骨髓基質(zhì)干細(xì)胞進(jìn)行實(shí)驗(yàn),實(shí)驗(yàn)分為四組：(1)BMSC培養(yǎng)組；(2)Ad-hBMP-2+BMSC培養(yǎng)組；(3)VEGF+BMSC培養(yǎng)組；(4)Ad-hBMP-2+VEGF+BMSC培養(yǎng)組。體外連續(xù)培養(yǎng)7天,分別在此期間內(nèi)用MTT法檢測(cè)各組細(xì)胞的增值能力,酶標(biāo)儀測(cè)定光密度值(OD值),每個(gè)樣本設(shè)六個(gè)復(fù)孔,求其平均值并繪制細(xì)胞生長(zhǎng)曲線。 4.低氧控制系統(tǒng)下,檢測(cè)轉(zhuǎn)染Ad-hBMP-2和VEGF的BMSC成骨能力 取第二代骨髓基質(zhì)干細(xì)胞進(jìn)行實(shí)驗(yàn),實(shí)驗(yàn)分為四組：(1)BMSC培養(yǎng)組；(2)Ad-hBMP-2+BMSC培養(yǎng)組；(3)VEGF+BMSC培養(yǎng)組；(4)Ad-hBMP-2+VEGF+BMSC培養(yǎng)組。96孔板內(nèi)于低氧控制系統(tǒng)下,連續(xù)誘導(dǎo)培養(yǎng)6天,分別在此6天內(nèi)用PNPP法檢測(cè)各組細(xì)胞的堿性磷酸酶(Alkaline Phosphatase,ALP)的活性的表達(dá)；酶標(biāo)儀測(cè)定光密度值(OD值),每個(gè)樣本設(shè)五個(gè)復(fù)孔,求其平均值,并繪制ALP活性增長(zhǎng)曲線；6孔板內(nèi)四組樣本體外連續(xù)誘導(dǎo)培養(yǎng)7天,在第7天行茜素紅染色,觀察礦化結(jié)節(jié)形成情況。 結(jié)果： 1.密度梯度離心法,可獲取高純度的BMSC,并且細(xì)胞活性較好。貼壁3天后細(xì)胞成長(zhǎng)為梭形及多角形,5、6天后增殖迅速,形成細(xì)胞集落。傳代后細(xì)胞形態(tài)更加單一,5、6天后即可鋪滿瓶底。 2.體外實(shí)驗(yàn)轉(zhuǎn)染骨髓基質(zhì)干細(xì)胞。Ad-GFP轉(zhuǎn)染12小時(shí)后即可檢測(cè)到綠色熒光蛋白表達(dá),轉(zhuǎn)染2天后平均轉(zhuǎn)染效率為90%,傳至5代后依然可檢測(cè)到綠色熒光蛋白表達(dá)。 3.MTT法檢測(cè)低氧控制系統(tǒng)下,轉(zhuǎn)染Ad-hBMP-2和Ad-VEGF的BMSC細(xì)胞增值及活力顯示：第1、2天各組細(xì)胞數(shù)量沒有明顯增加,各組間細(xì)胞增殖能力統(tǒng)計(jì)學(xué)上沒有顯著性差異(P0.05)；第3天后,各組細(xì)胞數(shù)量明顯增加,轉(zhuǎn)染Ad-hBMP-2的細(xì)胞增殖能力在統(tǒng)計(jì)學(xué)上顯著高于其他培養(yǎng)組(P0.01),轉(zhuǎn)染Ad-VEGF細(xì)胞增殖能力與空白組無顯著性差別(P0.05)。 4.低氧控制系統(tǒng)下,檢測(cè)轉(zhuǎn)染Ad-hBMP-2和VEGF的BMSC成骨能力 1)PNPP法檢測(cè)各組ALP活性表達(dá),結(jié)果顯示：Ad-hBMP-2+VEGF+BMSC培養(yǎng)組細(xì)胞成骨誘導(dǎo)培養(yǎng)后,ALP活性表達(dá)統(tǒng)計(jì)學(xué)上顯著高于其他三組(P0.05);Ad-hBMP-2+BMSC成骨誘導(dǎo)培養(yǎng)組高于BMSC組和VEGF+BMSC組(P0.05)；BMSC組和VEGF+BMSC組無顯著差別(P0.05)。 2)低氧控制系統(tǒng)下,4組樣本培養(yǎng)7天后,茜素紅染色結(jié)果顯示：Ad-hBMP-2+VEGF+BMSC成骨誘導(dǎo)培養(yǎng)組細(xì)胞密集生長(zhǎng)區(qū)有多量礦化結(jié)節(jié)形成,茜素紅染色成橘紅色。Ad-hBMP-2+BMSC和VEGF+BMSC成骨誘導(dǎo)培養(yǎng)組細(xì)胞有少量礦化結(jié)節(jié)。BMSC成骨誘導(dǎo)培養(yǎng)組只有零星幾個(gè)礦化結(jié)節(jié)。 結(jié)論： 在一周的培養(yǎng)時(shí)間內(nèi),骨形態(tài)形成蛋白(Bone Morphogenetic Protein2,BMP2)可以增強(qiáng)骨髓基質(zhì)干細(xì)胞(BMSC)的成骨活性；血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor, VEGF)短期內(nèi)對(duì)BMSC成骨活性影響和對(duì)照組無統(tǒng)計(jì)學(xué)差異；BMP-2和VEGF聯(lián)合作用比單獨(dú)轉(zhuǎn)染BMP-2更能顯著提高BMSC的成骨活性。
[Abstract]:Objective:
Bone morphogenetic protein (Bone Morphogenetic Protein 2, BMP2) is known to be the strongest promoter of ectopic osteogenesis, and vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) can significantly promote vascular regeneration and promote the secretion of healing.BMP2 and VEGF two proteins, which are beneficial to bone defects. The healing of bone defects, especially at the early stage of the healing process, is carried out in the hypoxic environment. Bone Marrow Stromal Cells (BMSC) has the potential of multiple cell differentiation to bone morphogenetic cells and angiogenic cells. This topic is to simulate the simultaneous transfection of B to hBMP2 and VEGF genes in hypoxic environment. MSC related characteristics in osteogenesis.
Method:
1. rat bone marrow stromal cells (BMSC) were isolated and cultured with Percoll separation solution (relative density 1.077). The bone marrow stromal stem cells were isolated from rat bone marrow by density gradient centrifugation, and were cultured and amplified in vitro.
2. the changes in the biological behavior and the expression of the target genes after BMSC transfection of hBMP-2 adenovirus vector (Ad-hBMP-2) and VEGF adenovirus vector (Ad-VEGF) were observed by morphological observation and determination of growth curve and Elisa.
After BMSC transfected to the adenovirus vector (Ad-GFP) of Green Fluorescent Protein (GFP), the transfection efficiency of adenovirus was measured by fluorescence microscope, and the expression of green fluorescent protein after transfection of Ad-GFP was observed.
3.MTT assay was used to detect the proliferation and viability of BMSC cells transfected with Ad-hBMP-2 and VEGF under hypoxia control system.
Second generations of bone marrow stromal stem cells were taken and divided into four groups: (1) BMSC culture group; (2) Ad-hBMP-2+BMSC culture group; (3) VEGF+BMSC culture group; (4) Ad-hBMP-2+VEGF+BMSC culture group. Continuous culture for 7 days in vitro, MTT method was used to detect the increment of cells in each group, and the enzyme meter measured light density value (o value), each The sample has six holes, and the average value is drawn and the cell growth curve is drawn.
4. osteogenic ability of BMSC transfected with Ad-hBMP-2 and VEGF under hypoxia control system
Second generations of bone marrow stromal cells were taken and divided into four groups: (1) BMSC culture group; (2) Ad-hBMP-2+BMSC culture group; (3) VEGF+BMSC culture group; (4) Ad-hBMP-2+VEGF+BMSC culture group in.96 orifice plate under hypoxia control system for 6 days for 6 days, and the PNPP method was used to detect alkaline phosphatase in each group of cells (Alk) within this 6 day (Alk The expression of the activity of aline Phosphatase, ALP); the light density value (o value) was measured by the enzyme labeled instrument. Five compound holes were set in each sample, and the average value of each sample was set, and the growth curve of ALP activity was drawn. The four groups of samples in the 6 hole plate were continuously induced and cultured for 7 days in vitro, and the seventh days were stained with alizarin red, and the formation of mineralized nodules was observed.
Result:
The 1. density gradient centrifugation method can obtain high purity BMSC, and the cell activity is good. Cells grow into spindle and polygon after 3 days. After 5,6 days, the cells proliferate rapidly and form cell colonies. After the passage, the cell morphology is more single and can be covered with the bottom of the bottle after 5,6 days.
2. the expression of green fluorescent protein could be detected after transfection of bone marrow stromal stem cells.Ad-GFP in vitro for 12 hours. The average transfection efficiency was 90% after 2 days of transfection, and the expression of green fluorescent protein could still be detected after 5 generations.
The proliferation and vitality of BMSC cells transfected with 3.MTT and Ad-VEGF showed that the number of BMSC cells transfected with Ad-hBMP-2 and Ad-VEGF showed no significant increase in number of cells on day 1,2, and there was no significant difference in cell proliferation between each group (P0.05). After third days, the number of cells in each group increased significantly, and the proliferation ability of Ad-hBMP-2 transfected to Ad-hBMP-2 Compared with other culture groups (P0.01), the proliferation of Ad-VEGF cells was not significantly different from that of the blank group (P0.05).
4. osteogenic ability of BMSC transfected with Ad-hBMP-2 and VEGF under hypoxia control system
1) the expression of ALP activity in each group was detected by PNPP method. The results showed that the expression of ALP activity was significantly higher than that of the other three groups (P0.05) after the induction of osteogenesis in the Ad-hBMP-2+VEGF+BMSC culture group, and the Ad-hBMP-2+BMSC osteogenic induction group was higher than the BMSC group and VEGF+BMSC group (P0.05), and there was no significant difference between the BMSC group and the VEGF+BMSC group (P0.05).
2) under the hypoxic control system, 4 groups of samples were cultured for 7 days. The results of alizarin red staining showed that there were many mineralized nodules in the cell intensive growth area of Ad-hBMP-2+VEGF+BMSC osteogenesis induced culture group. Alizarin red was stained into orange red.Ad-hBMP-2+BMSC and VEGF+BMSC induction culture group with a small amount of mineralized nodules in.BMSC osteogenic induction culture group. There are sporadic mineralized nodules.
Conclusion:
Bone morphogenetic protein (Bone Morphogenetic Protein2, BMP2) can enhance the osteogenic activity of bone marrow stromal cells (BMSC) in a week of culture, and there is no statistical difference between the vascular endothelial growth factor (VEGF) and the control group in the short term. Compared with the transfection of BMP-2 alone, the osteogenic activity of BMSC could be significantly improved.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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相關(guān)期刊論文 前1條

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本文編號(hào)：2096728