發(fā)布時(shí)間：2018-05-10 15:04

  本文選題：急性心肌梗死 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《蘇州大學(xué)》2008年碩士論文

【摘要】： 目的:骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenchyme stem cell,BMSCs)來(lái)源于中胚層,具有自我更新和多向分化潛能,可以體外分離培養(yǎng),經(jīng)一定條件誘導(dǎo),可以向心肌細(xì)胞方向分化。5-氮胞苷(5-azacytidine,5-aza)誘導(dǎo)后,可使得某些基因的肌源性決定部位去甲基化,轉(zhuǎn)錄激活,引起細(xì)胞向肌源性細(xì)胞分化。成纖維細(xì)胞生長(zhǎng)因子-2(FGF-2)是一種有絲分裂原,具有促進(jìn)細(xì)胞分裂增殖的效應(yīng)。據(jù)報(bào)道,此因子還具有心臟保護(hù)作用,可以誘導(dǎo)心肌肥大,并且可以提高心肌細(xì)胞在缺氧條件下的存活率。超聲介導(dǎo)的白蛋白微泡破裂可以促進(jìn)DNA分子穿透細(xì)胞膜和核膜并整合入細(xì)胞基因組中。本研究目的:(1)體外克隆FGF-2基因,重組并構(gòu)建其真核表達(dá)載體;(2)探討B(tài)MSCs體外分離培養(yǎng)并誘導(dǎo)其向心肌細(xì)胞分化方法;(3)探討超聲介導(dǎo)白蛋白微泡破裂促進(jìn)FGF-2轉(zhuǎn)移BMSCs的方法,并和脂質(zhì)體(lipsome2000)介導(dǎo)轉(zhuǎn)移BMSCs相比較。 方法:(1)采用全骨髓培養(yǎng)法和Ficoll分離法體外分離培養(yǎng)大鼠BMSCs,觀察其生長(zhǎng)特性,表面抗原表達(dá)等,HE染色觀察BMSCs細(xì)胞形態(tài),胞核和胞漿,MTT法分別測(cè)定誘導(dǎo)和未誘導(dǎo)BMSCs的生長(zhǎng)曲線。在第4代加入10gmol/L 5-aza預(yù)誘導(dǎo)BMSCs24小時(shí)(hr),第7代再一次加入10pμmol/L 5-aza誘導(dǎo)24hr;流式細(xì)胞儀技術(shù)、免疫熒光及RT-PCR實(shí)驗(yàn)對(duì)誘導(dǎo)前后的BMSCs進(jìn)行表型鑒定;(2)分離培養(yǎng)新生大鼠心室肌細(xì)胞,觀察其體外生長(zhǎng)的形態(tài)、特性,以及抗原表達(dá)等特征:(3)收集體外培養(yǎng)的心室肌細(xì)胞,Trizol法提取心室肌細(xì)胞總mRNA,RT-PCR克隆擴(kuò)增大鼠FGF-2基因,與PIRES2-EGFP載體連接,構(gòu)建重組PIRES2-EGFP-FGF-2真核表達(dá)載體;(4)調(diào)整超聲強(qiáng)度和作用時(shí)間,尋找最佳作用條件,在超聲介導(dǎo)白蛋白微泡破裂作用下促進(jìn)FGF-2基因轉(zhuǎn)移BMSCs,24hr后熒光顯微鏡下觀察EGFP表達(dá)情況;(5)在一定劑量的lipsome2000作用下,促進(jìn)FGF-2基因轉(zhuǎn)移BMSCs:(6)臺(tái)盼藍(lán)染色法測(cè)定不同超聲強(qiáng)度不同作用時(shí)間之后BMSCs的活性。(7)流式細(xì)胞儀技術(shù)檢測(cè)超聲組和脂質(zhì)體組轉(zhuǎn)移FGF-2之后報(bào)告基因EGFP的表達(dá)。 結(jié)果:(1)由新生大鼠心室肌組織分離培養(yǎng)得到心室肌細(xì)胞呈簇樣生長(zhǎng)并重疊成致密有邊界的細(xì)胞團(tuán),培養(yǎng)10天左右,倒置相差顯微鏡下可見(jiàn)成團(tuán)的心室肌細(xì)胞出現(xiàn)節(jié)律性收縮,收縮時(shí)間長(zhǎng)達(dá)半個(gè)月之久。 (2)從心室肌細(xì)胞中擴(kuò)增FGF-2基因,構(gòu)建重組質(zhì)粒PIRES2-EGFP-FGF-2,測(cè)序正確的陽(yáng)性克隆保種備用。 (3)由骨髓分離得到的BMSCs體外培養(yǎng)呈長(zhǎng)梭形貼壁生長(zhǎng),部分造血細(xì)胞懸浮在培養(yǎng)基中,MTT生長(zhǎng)曲線顯示,細(xì)胞在接種后第2天即進(jìn)入對(duì)數(shù)生長(zhǎng)期,細(xì)胞增殖活躍,倒置相差顯微鏡下觀察細(xì)胞向外周伸展,兩端有軸狀突起,細(xì)胞呈長(zhǎng)梭形,有雙核分裂相的BMSCs多見(jiàn),胞核較圓,核質(zhì)豐富,核仁可見(jiàn)。隨著細(xì)胞生長(zhǎng)密度增大,彼此相連;7天可鋪滿瓶底,生長(zhǎng)迅速,可大量繁殖,之后如不傳代,則有老化現(xiàn)象。12天左右,BMSCs生長(zhǎng)緩慢,進(jìn)入平臺(tái)期。 (4)運(yùn)用免疫熒光和流式細(xì)胞儀技術(shù)檢測(cè)顯示該群細(xì)胞高表達(dá)CD44(99.3%),CD54(97.7%),CD90(99.4%),CD71(98.2%),CD106(25.8%)表達(dá)相對(duì)較低,低表達(dá)CD45(8.9%),CD31(15.9%)等造血細(xì)胞表面標(biāo)志。BMSCs經(jīng)傳代之后,細(xì)胞形態(tài)比較均一,生長(zhǎng)速度較快,平均3-4d可鋪滿瓶底,細(xì)胞接種密度較大,接觸抑制比較明顯。第10代BMSCs HE染色,細(xì)胞仍呈長(zhǎng)梭形,兩端有軸狀突起,胞漿豐富深染成紅色,胞核圓形或橢圓形,呈藍(lán)色,核仁明顯。 (5)BMSCs經(jīng)5-aza誘導(dǎo)5天后,倒置相差顯微鏡下觀察,細(xì)胞呈集落樣生長(zhǎng),體積變大變圓,大小不等,核質(zhì)豐富深染,比較粗糙,胞核較圓,核仁清楚,多分裂相,增殖能力強(qiáng),細(xì)胞之間互相交聯(lián),緊密連接。免疫熒光染色后發(fā)現(xiàn)經(jīng)5-aza誘導(dǎo)后的BMSCs可表達(dá)心肌細(xì)胞特異性分子肌球蛋白重鏈(MHC-β)、連接蛋白43(connexin43)、結(jié)蛋白(desmin)以及橫紋肌肌動(dòng)蛋白(actinin-α);而部分經(jīng)誘導(dǎo)的細(xì)胞也表達(dá)血管內(nèi)皮細(xì)胞特異性分子(Flk-1/VEGFR2/KDR)。經(jīng)RT-PCR檢測(cè),誘導(dǎo)的BMSCs表達(dá)心肌細(xì)胞早期發(fā)育基因NKX2.5和GATA4。 (6)FGF-2基因在超聲介導(dǎo)白蛋白微泡破裂作用下轉(zhuǎn)移到BMSCs。熒光顯微鏡下可看到綠色熒光,細(xì)胞表達(dá)EGFP;lipsome2000介導(dǎo)的FGF-2基因轉(zhuǎn)移大鼠BMSCs,24hr后熒光顯微鏡下觀察,可見(jiàn)到綠色熒光,細(xì)胞表達(dá)EGFP,FGF-2基因轉(zhuǎn)移BMSCs中。 (7)不同超聲強(qiáng)度和作用時(shí)間對(duì)于BMSCs活力影響不同,通過(guò)測(cè)定,在不超過(guò)0.75W/cm~2的超聲強(qiáng)度,32sec的作用時(shí)間之內(nèi),對(duì)于細(xì)胞都是安全的。0.75W/cm~2為最好轉(zhuǎn)移強(qiáng)度,32sec為最好轉(zhuǎn)移時(shí)間。 (8)FGF-2轉(zhuǎn)移BMSCs 48hr后進(jìn)行流式細(xì)胞儀技術(shù)測(cè)定報(bào)告基因EGFP的表達(dá),脂質(zhì)體組報(bào)告基因EGFP表達(dá)率(49.35a±9.34)%,明顯高于超聲組(30.42±4.58)%,有統(tǒng)計(jì)學(xué)差異(P＜0.05);脂質(zhì)體組轉(zhuǎn)移效率高于超聲組。 結(jié)論:(1)采用組織塊培養(yǎng)法,可體外成功分離培養(yǎng)新生Sprague-Dawley(SD)大鼠心室肌細(xì)胞,采用免疫熒光方法,可對(duì)表面抗原進(jìn)行鑒定。 (2)收集SD大鼠心室肌細(xì)胞,Trizol法抽提RNA,依照NCBI Genebank中大鼠FGF-2基因序列,設(shè)計(jì)適當(dāng)引物,可克隆出大鼠FGF-2基因,并成功構(gòu)建了其真核表達(dá)載體。 (3)一定濃度的5-aza可誘導(dǎo)BMSCs向心肌細(xì)胞分化。 (4)不同超聲強(qiáng)度、不同作用時(shí)間對(duì)于BMSCs活力影響不同。 (5)脂質(zhì)體和超聲介導(dǎo)的白蛋白微泡破裂均可以促進(jìn)FGF-2轉(zhuǎn)移BMSCs。 (6)超聲介導(dǎo)白蛋白微泡破裂可促進(jìn)FGF-2轉(zhuǎn)移BMSCs,為非病毒載體進(jìn)行基因轉(zhuǎn)移提供了新的方法,但轉(zhuǎn)移效率有待進(jìn)一步提高。
[Abstract]:Objective: Bone marrow mesenchyme stem cell (BMSCs) is derived from the mesoderm, which has the potential of self renewal and multidirectional differentiation. It can be isolated and cultured in vitro, and can be induced by certain conditions. After the induction of.5- azacytidine (5-azacytidine, 5-aza) in the direction of cardiac myocyte, it can make certain genes in the myogenic location. Demethylation, transcriptional activation, causes cells to differentiate into myogenic cells. Fibroblast growth factor -2 (FGF-2) is a mitogen that promotes cell division and proliferation. It is reported that this factor also has a protective effect on heart, can induce cardiac hypertrophy, and can increase the survival rate of cardiac myocytes under hypoxia. Ultrasound mediated albumin microbubble rupture can promote DNA molecules to penetrate cell membranes and nuclear membranes and integrate into the cell genome. (1) the FGF-2 gene was cloned in vitro to restructure and construct its eukaryotic expression vector; (2) to explore the isolation and culture of BMSCs in vitro and to induce its differentiation into cardiomyocytes; (3) to explore ultrasound mediated albumin micro Bubble rupture promotes the transfer of BMSCs by FGF-2 and is compared with liposome (lipsome2000) mediated BMSCs transfer.
Methods: (1) BMSCs was isolated and cultured in vitro by full bone marrow culture and Ficoll separation in vitro. The growth characteristics, surface antigen expression, BMSCs cell morphology, nucleus and cytoplasm were observed by HE staining. The growth curves of induced and uninduced BMSCs were measured by MTT method respectively. The pre induced BMSCs24 hours (HR) and the seventh generation after the addition of 10gmol/L 5-aza in the fourth generation. One time 10p micron mol/L 5-aza was added to induce 24hr; flow cytometry, immunofluorescence and RT-PCR experiments were used to identify the phenotype of BMSCs before and after induction. (2) the isolated and cultured neonatal rat ventricular myocytes were isolated and cultured in vitro, and observed the morphology, characteristics and antigen expression in vitro: (3) collecting the cultured ventricular myocytes and Trizol method to extract the heart. The total mRNA, RT-PCR cloned and amplified FGF-2 gene of rat, connected with PIRES2-EGFP vector, and constructed the recombinant PIRES2-EGFP-FGF-2 eukaryotic expression vector; (4) adjust the ultrasonic intensity and time to find the best conditions, promote the FGF-2 gene transfer BMSCs under the action of ultrasound mediated albumin microbubble rupture, and observe E under 24hr after the fluorescence microscope. The expression of GFP; (5) under the action of a certain dose of lipsome2000, FGF-2 gene transfer BMSCs: (6) trypan blue staining method was used to determine the activity of BMSCs after different ultrasonic intensity different action time. (7) flow cytometry was used to detect the expression of the reporter gene EGFP in the ultrasound group and the liposome group after the transfer of FGF-2.
Results: (1) the ventricular myocytes of the newborn rats were isolated and cultured, and the ventricular myocytes were clustered and overlapped into a dense and boundary cell group. The cells were cultured for about 10 days. Under the inverted phase contrast microscope, the ventricular myocytes of the ventricular myocytes appeared rhythmical contraction for up to half a month.
(2) to amplify the FGF-2 gene from ventricular myocytes, construct the recombinant plasmid PIRES2-EGFP-FGF-2, and sequenced the positive clones for preservation.
(3) the culture of BMSCs in vitro was long shuttle shaped and adhered to the wall, and some of the hematopoietic cells were suspended in the medium. The MTT growth curve showed that the cells entered the logarithmic growth period after second days of inoculation, the cell proliferation was active, and the cells spread to the periphery under the inverted phase contrast microscope, the two ends had axial protuberances, and the cells showed a long shuttle shape. The BMSCs of the mitotic phase is more common, the nucleus is more round, the nucleolus is rich and the nucleolus can be seen. With the growth density of the cells, it is connected with each other; 7 days can be filled with the bottom of the bottle. It can grow rapidly and can reproduce a lot. Then, if no generation is passed, the aging phenomenon is about.12 days, the growth of BMSCs is slow and into platform period.
(4) immunofluorescence and flow cytometry were used to detect the high expression of CD44 (99.3%), CD54 (97.7%), CD90 (99.4%), CD71 (98.2%), CD106 (25.8%), low expression of CD45 (8.9%), CD31 (15.9%) and other hematopoietic cell surface markers, after the passage of.BMSCs, the cell morphology was relatively uniform, the growth rate was faster, and the average 3-4d could be spread. At the bottom of the bottle, the cell inoculation density is larger and the contact inhibition is obvious. The tenth generation BMSCs HE staining, the cells still have long spindle shape, the two ends have axial protuberance, the cytoplasm is deep dyed red, the nucleus is round or oval, blue and the nucleolus are obvious.
(5) BMSCs was induced by 5-aza after 5 days, and under the inverted phase contrast microscope, the cells showed colony like growth, the volume became large and round, the size was different, the nuclear substance was rich and deep dye, the nucleus was round, the nucleolus was clear, the proliferation was strong, the cells cross linked and connected closely. After immunofluorescence staining, the BMSCs induced by 5-aza could be found. Expression of specific molecular myosin heavy chain (MHC- beta), connexin 43 (connexin43), protein (desmin) and rhabdomyosin actin (actinin- alpha), while some of the induced cells also express vascular endothelial cell specific molecule (Flk-1/VEGFR2/KDR). RT-PCR detection, induced BMSCs to express the early development gene N of cardiac myocyte KX2.5 and GATA4.
(6) FGF-2 gene can be transferred to BMSCs. fluorescence microscope under the action of ultrasound mediated albumin microbubble rupture to see green fluorescence, cell expression EGFP; lipsome2000 mediated FGF-2 gene transfer rat BMSCs, 24hr after 24hr fluorescence microscope observation, can see green fluorescence, cell expression EGFP, FGF-2 gene transfer BMSCs.
(7) different ultrasonic intensity and action time have different effects on BMSCs activity. By measuring, the best transfer intensity is.0.75W/cm~2 for the cell is safe and the.0.75W/cm~2 is the best transfer time, and 32sec is the best transfer time without more than 0.75W/cm~2 ultrasonic intensity and 32sec action time.
(8) after FGF-2 transfer of BMSCs 48hr, the expression of the reporter gene EGFP was detected by flow cytometry, and the EGFP expression rate of the liposome group was (49.35a + 9.34)%, which was significantly higher than that in the ultrasound group (30.42 + 4.58)% (P < 0.05), and the transfer efficiency of the liposome group was higher than that of the ultrasound group.
Conclusion: (1) the ventricular myocytes of neonatal Sprague-Dawley (SD) rats can be successfully isolated and cultured in vitro by tissue mass culture, and the immunofluorescence method can be used to identify the surface antigens.
(2) SD rat ventricular myocytes were collected and RNA was extracted by Trizol. According to the sequence of FGF-2 gene in NCBI Genebank, appropriate primers were designed to clone the rat FGF-2 gene, and the eukaryotic expression vector was successfully constructed.
(3) a certain concentration of 5-aza can induce BMSCs to differentiate into cardiomyocytes.
(4) different ultrasound intensity and different action time had different effects on BMSCs activity.
(5) liposomes and ultrasound mediated disruption of albumin microbubbles can promote FGF-2 metastasis to BMSCs.
(6) ultrasound mediated albumin microbubble rupture can promote FGF-2 transfer of BMSCs, which provides a new method for non viral vector gene transfer, but the transfer efficiency needs to be further improved.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

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