超声介导白蛋白微泡破裂促进成纤维细胞生长因子-2转移骨髓间充质干细胞的实验研究

发布时间：2018-05-10 15:04

本文选题：急性心肌梗死 + 骨髓间充质干细胞　；参考：《苏州大学》2008年硕士论文

【摘要】： 目的:骨髓间充质干细胞(Bone marrow mesenchyme stem cell,BMSCs)来源于中胚层,具有自我更新和多向分化潜能,可以体外分离培养,经一定条件诱导,可以向心肌细胞方向分化。5-氮胞苷(5-azacytidine,5-aza)诱导后,可使得某些基因的肌源性决定部位去甲基化,转录激活,引起细胞向肌源性细胞分化。成纤维细胞生长因子-2(FGF-2)是一种有丝分裂原,具有促进细胞分裂增殖的效应。据报道,此因子还具有心脏保护作用,可以诱导心肌肥大,并且可以提高心肌细胞在缺氧条件下的存活率。超声介导的白蛋白微泡破裂可以促进DNA分子穿透细胞膜和核膜并整合入细胞基因组中。本研究目的:(1)体外克隆FGF-2基因,重组并构建其真核表达载体;(2)探讨BMSCs体外分离培养并诱导其向心肌细胞分化方法;(3)探讨超声介导白蛋白微泡破裂促进FGF-2转移BMSCs的方法,并和脂质体(lipsome2000)介导转移BMSCs相比较。方法:(1)采用全骨髓培养法和Ficoll分离法体外分离培养大鼠BMSCs,观察其生长特性,表面抗原表达等,HE染色观察BMSCs细胞形态,胞核和胞浆,MTT法分别测定诱导和未诱导BMSCs的生长曲线。在第4代加入10gmol/L 5-aza预诱导BMSCs24小时(hr),第7代再一次加入10pμmol/L 5-aza诱导24hr;流式细胞仪技术、免疫荧光及RT-PCR实验对诱导前后的BMSCs进行表型鉴定;(2)分离培养新生大鼠心室肌细胞,观察其体外生长的形态、特性,以及抗原表达等特征:(3)收集体外培养的心室肌细胞,Trizol法提取心室肌细胞总mRNA,RT-PCR克隆扩增大鼠FGF-2基因,与PIRES2-EGFP载体连接,构建重组PIRES2-EGFP-FGF-2真核表达载体;(4)调整超声强度和作用时间,寻找最佳作用条件,在超声介导白蛋白微泡破裂作用下促进FGF-2基因转移BMSCs,24hr后荧光显微镜下观察EGFP表达情况;(5)在一定剂量的lipsome2000作用下,促进FGF-2基因转移BMSCs:(6)台盼蓝染色法测定不同超声强度不同作用时间之后BMSCs的活性。(7)流式细胞仪技术检测超声组和脂质体组转移FGF-2之后报告基因EGFP的表达。结果:(1)由新生大鼠心室肌组织分离培养得到心室肌细胞呈簇样生长并重叠成致密有边界的细胞团,培养10天左右,倒置相差显微镜下可见成团的心室肌细胞出现节律性收缩,收缩时间长达半个月之久。 (2)从心室肌细胞中扩增FGF-2基因,构建重组质粒PIRES2-EGFP-FGF-2,测序正确的阳性克隆保种备用。 (3)由骨髓分离得到的BMSCs体外培养呈长梭形贴壁生长,部分造血细胞悬浮在培养基中,MTT生长曲线显示,细胞在接种后第2天即进入对数生长期,细胞增殖活跃,倒置相差显微镜下观察细胞向外周伸展,两端有轴状突起,细胞呈长梭形,有双核分裂相的BMSCs多见,胞核较圆,核质丰富,核仁可见。随着细胞生长密度增大,彼此相连;7天可铺满瓶底,生长迅速,可大量繁殖,之后如不传代,则有老化现象。12天左右,BMSCs生长缓慢,进入平台期。 (4)运用免疫荧光和流式细胞仪技术检测显示该群细胞高表达CD44(99.3%),CD54(97.7%),CD90(99.4%),CD71(98.2%),CD106(25.8%)表达相对较低,低表达CD45(8.9%),CD31(15.9%)等造血细胞表面标志。BMSCs经传代之后,细胞形态比较均一,生长速度较快,平均3-4d可铺满瓶底,细胞接种密度较大,接触抑制比较明显。第10代BMSCs HE染色,细胞仍呈长梭形,两端有轴状突起,胞浆丰富深染成红色,胞核圆形或椭圆形,呈蓝色,核仁明显。 (5)BMSCs经5-aza诱导5天后,倒置相差显微镜下观察,细胞呈集落样生长,体积变大变圆,大小不等,核质丰富深染,比较粗糙,胞核较圆,核仁清楚,多分裂相,增殖能力强,细胞之间互相交联,紧密连接。免疫荧光染色后发现经5-aza诱导后的BMSCs可表达心肌细胞特异性分子肌球蛋白重链(MHC-β)、连接蛋白43(connexin43)、结蛋白(desmin)以及横纹肌肌动蛋白(actinin-α);而部分经诱导的细胞也表达血管内皮细胞特异性分子(Flk-1/VEGFR2/KDR)。经RT-PCR检测,诱导的BMSCs表达心肌细胞早期发育基因NKX2.5和GATA4。 (6)FGF-2基因在超声介导白蛋白微泡破裂作用下转移到BMSCs。荧光显微镜下可看到绿色荧光,细胞表达EGFP;lipsome2000介导的FGF-2基因转移大鼠BMSCs,24hr后荧光显微镜下观察,可见到绿色荧光,细胞表达EGFP,FGF-2基因转移BMSCs中。 (7)不同超声强度和作用时间对于BMSCs活力影响不同,通过测定,在不超过0.75W/cm~2的超声强度,32sec的作用时间之内,对于细胞都是安全的。0.75W/cm~2为最好转移强度,32sec为最好转移时间。 (8)FGF-2转移BMSCs 48hr后进行流式细胞仪技术测定报告基因EGFP的表达,脂质体组报告基因EGFP表达率(49.35a±9.34)%,明显高于超声组(30.42±4.58)%,有统计学差异(P＜0.05);脂质体组转移效率高于超声组。结论:(1)采用组织块培养法,可体外成功分离培养新生Sprague-Dawley(SD)大鼠心室肌细胞,采用免疫荧光方法,可对表面抗原进行鉴定。 (2)收集SD大鼠心室肌细胞,Trizol法抽提RNA,依照NCBI Genebank中大鼠FGF-2基因序列,设计适当引物,可克隆出大鼠FGF-2基因,并成功构建了其真核表达载体。 (3)一定浓度的5-aza可诱导BMSCs向心肌细胞分化。 (4)不同超声强度、不同作用时间对于BMSCs活力影响不同。 (5)脂质体和超声介导的白蛋白微泡破裂均可以促进FGF-2转移BMSCs。 (6)超声介导白蛋白微泡破裂可促进FGF-2转移BMSCs,为非病毒载体进行基因转移提供了新的方法,但转移效率有待进一步提高。
[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.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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