本文選題：成骨細(xì)胞 + 血管內(nèi)皮細(xì)胞��； 參考：《瀘州醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的：近年來(lái)，組織工程骨已成為社會(huì)研究的熱點(diǎn)，而成骨細(xì)胞和血管內(nèi)皮細(xì)胞共培養(yǎng)是組織工程骨研究的首要條件。本實(shí)驗(yàn)將從共培養(yǎng)體系的建立入手，進(jìn)而研究共培養(yǎng)體系對(duì)胰島素生長(zhǎng)因子1（insulin-like growthfactor-1,IGF-1）表達(dá)的影響及對(duì)血管內(nèi)皮細(xì)胞生物學(xué)特性的影響。方法：本實(shí)驗(yàn)分為三部分，第一部分以成骨樣肉瘤細(xì)胞株（Human osteosarcoma MG-63cell, MG63）及血管內(nèi)皮細(xì)胞株（Vascularendothelial cells, VECs）為研究對(duì)象，細(xì)胞傳代培養(yǎng)至第六代備用，通過(guò)5種不同的比例建立MG63-VECs共培養(yǎng)體系，通過(guò)倒置顯微鏡下細(xì)胞形態(tài)的觀(guān)察來(lái)確定兩種細(xì)胞的最佳共培養(yǎng)比例；第二部分，以第一部分的最佳共培養(yǎng)比例建立MG63-VECs共培養(yǎng)體系作為實(shí)驗(yàn)組，MG63及VECs單獨(dú)培養(yǎng)作為對(duì)照組，分別在12h,24h,36h及48h提取各組上清液，利用ELISA實(shí)驗(yàn)方法對(duì)實(shí)驗(yàn)組和對(duì)照組各個(gè)時(shí)間點(diǎn)進(jìn)行IGF-1表達(dá)值的檢測(cè)；再利用Transwell小室將VECs及MG63按最佳比例進(jìn)行間接共培養(yǎng)，實(shí)驗(yàn)分為4組，共培養(yǎng)A組，共培養(yǎng)B組，MG63組，VECs組，分別在12h,24h,36h及48h對(duì)各組進(jìn)行RNA提取，再用熒光定量PCR檢測(cè)IGF-1mRNA的表達(dá)。第三部分觀(guān)察共培養(yǎng)體系對(duì)血管內(nèi)皮細(xì)胞生物學(xué)特性的影響，一方面通過(guò)DIi染色示蹤法觀(guān)察，將VECs進(jìn)行DIi染色，與MG63按1：1進(jìn)行直接共培養(yǎng)為實(shí)驗(yàn)組，VECs進(jìn)行DIi染色后單獨(dú)培養(yǎng)為對(duì)照組，96h后在熒光顯微鏡下觀(guān)察兩組的管型情況；另一方面通過(guò)鼠尾膠原實(shí)驗(yàn)觀(guān)察。利用鼠尾膠原制備三維培養(yǎng)環(huán)境，實(shí)驗(yàn)組為MG63與VECs按1：1進(jìn)行直接共培養(yǎng)，對(duì)照組為VECs單獨(dú)培養(yǎng)。72h后在倒置顯微鏡下觀(guān)察管型的情況。以上原始實(shí)驗(yàn)數(shù)據(jù)均使用SPSS13.0軟件分析，數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差（x±s）表示，兩樣本均數(shù)比較用獨(dú)立樣本t檢驗(yàn)；多個(gè)樣本兩兩比較先用方差分析（ANOVA），再用多樣本兩兩比較中的SNK法進(jìn)行比較，p0.05及p0.01表示差異有顯著性和非常顯著性意義。結(jié)果：第一部分，MG63培養(yǎng)至第五代在倒置顯微鏡下觀(guān)察，細(xì)胞形態(tài)不規(guī)則，多為三角形及多角形。VECs呈圓形或橢圓形，呈鋪路石樣生長(zhǎng)；直接共培養(yǎng)中MG63和VECs細(xì)胞按1:1培養(yǎng)時(shí)兩種細(xì)胞生長(zhǎng)最為良好，細(xì)胞間雜生長(zhǎng)，其中血管內(nèi)皮細(xì)胞呈結(jié)節(jié)樣生長(zhǎng)，成骨細(xì)胞生長(zhǎng)于血管內(nèi)皮細(xì)胞結(jié)節(jié)周?chē)�。第二部分，ELISA結(jié)果顯示：共培養(yǎng)組IGF-1表達(dá)值較對(duì)照組明顯增加(P0.05)。熒光定量PCR結(jié)果顯示：12h-48h共培養(yǎng)A組及MG63組IGF-1mRNA表達(dá)值隨著時(shí)間呈上升趨勢(shì)，共培養(yǎng)A組較MG63組IGF-1mRNA表達(dá)值明顯增加(P0.05)；共培養(yǎng)B組和VECs組IGF-1mRNA表達(dá)值隨著時(shí)間出現(xiàn)不穩(wěn)定變化趨勢(shì)，在12h和36h呈上升趨勢(shì)，，24h和48h時(shí)呈下降趨勢(shì)；共培養(yǎng)A組各時(shí)間點(diǎn)IGF-1mRNA表達(dá)值明顯高于共培養(yǎng)B組養(yǎng)(P0.01)。第三部分，體外管型實(shí)驗(yàn)，DIi示蹤法96h熒光顯微鏡下觀(guān)察實(shí)驗(yàn)組VECs出現(xiàn)類(lèi)管型樣結(jié)構(gòu)，對(duì)照組VECs上層未見(jiàn)明顯管型樣結(jié)構(gòu)形成。鼠尾膠實(shí)驗(yàn)72h倒置顯微鏡下觀(guān)察，對(duì)照組形成類(lèi)血管腔樣結(jié)構(gòu)；實(shí)驗(yàn)組少量細(xì)胞壞死，未見(jiàn)明顯的血管腔樣結(jié)構(gòu)形成。結(jié)論：1、直接共培養(yǎng)中MG63和VECs按1:1培養(yǎng)時(shí)兩種細(xì)胞生長(zhǎng)最為良好，細(xì)胞間雜生長(zhǎng)，其中VECs呈結(jié)節(jié)樣生長(zhǎng)，MG63生長(zhǎng)于VECs結(jié)節(jié)周?chē)?、在MG63-VECs共培養(yǎng)體系中IGF-1的表達(dá)值優(yōu)于二者的單獨(dú)培養(yǎng)；間接共培養(yǎng)條件下共培養(yǎng)A組IGF-1mRNA表達(dá)明顯高于共培養(yǎng)B組。3、通過(guò)共培養(yǎng)體系的管型實(shí)驗(yàn)證明在成骨細(xì)胞的參與下血管內(nèi)皮細(xì)胞更易形成管型驗(yàn)證了在骨組織血管化中成骨細(xì)胞不僅僅構(gòu)建了骨組織基本結(jié)構(gòu)，還作為一種管型誘導(dǎo)功能細(xì)胞，參與了VECs的遷移和管型形成。
[Abstract]:Objective: in recent years, tissue engineering bone has become a hot spot in social research, and co culture of osteoblasts and vascular endothelial cells is the primary condition for the study of tissue engineering bone. This experiment will start with the establishment of co culture system and then study the effect of co culture system on the expression of insulin growth factor 1 (insulin-like growthfactor-1, IGF-1). And the effect on biological characteristics of vascular endothelial cells. Methods: this experiment was divided into three parts. The first part was Human osteosarcoma MG-63cell (MG63) and vascular endothelial cell line (Vascularendothelial cells, VECs). The cells were cultured to sixth generations and were established by 5 different proportions. MG63-VECs co culture system, the optimum co culture ratio of two kinds of cells was determined by the observation of cell morphology under inverted microscope. The second part, using the best co culture ratio of the first part to establish the MG63-VECs co culture system as the experimental group. MG63 and VECs were cultured separately as the group, and respectively in 12h, 24h, 36h and 48h, respectively. The ELISA test method was used to detect the IGF-1 expression value at each time point of the experimental group and the control group. Then the Transwell chamber was used to direct the indirect co culture of VECs and MG63 according to the optimum proportion. The experiment was divided into 4 groups. The experiment was divided into groups of A groups, and a total of B, MG63, VECs groups were cultured in the B, MG63, and VECs groups. The expression of IGF-1mRNA was detected by quantitative PCR. The third part observed the effect of co culture system on the biological characteristics of vascular endothelial cells. On the one hand, the VECs was stained by DIi staining, and MG63 was directly co cultured with 1:1 as the experimental group. VECs was stained by DIi to be the control group, and 96h after the fluorescence microscope. On the other hand, the tube type of the two groups was observed. On the other hand, the rat tail collagen was observed. The three-dimensional culture environment was prepared by the rat tail collagen. The experimental group was co cultured with MG63 and VECs according to 1:1. The control group observed the tube type under the inverted microscope after the single culture of VECs by the inverted microscope. All the above original experimental data were divided into SPSS13.0 software. Analysis, the data was expressed with mean mean standard deviation (x + s). The average number of two samples was compared with independent sample t test; 22 samples were compared with variance analysis (ANOVA) first, and then compared with SNK in multi sample 22 comparison. P0.05 and P0.01 showed significant and significant difference. The first part, MG63 was cultivated to fifth generations. Under the inverted microscope, the morphology of the cells was irregular, and the.VECs was round or oval in the shape of triangle and polygon. The growth of the two cells was the best in the direct co culture of MG63 and VECs cells, and the cell growth was nodular, and the osteoblasts grew in the blood vessels. The second part of the endothelial cell nodules. The results of ELISA showed that the expression of IGF-1 in the co culture group was significantly higher than that in the control group (P0.05). The results of fluorescence quantitative PCR showed that the IGF-1mRNA expression value of the 12h-48h co culture A group and the MG63 group increased with the time, and the co culture A group was significantly higher than the MG63 group IGF-1mRNA expression value (P0.05). The expression value of IGF-1mRNA in group VECs and VECs showed a trend of unstable change along with time, which showed an upward trend in 12h and 36h, and decreased in 24h and 48h. The IGF-1mRNA expression value at each time point in the co culture A group was obviously higher than that in co culture B group (P0.01). The third part, in vitro tube type experiment, DIi tracing method under 96h fluorescence microscope, observed the occurrence class of experimental group. In the control group, there was no obvious tube like structure in the control group. The rat tail gum was observed under 72h inverted microscope and the control group formed a blood vessel like structure. In the experimental group, a small number of cells were necrotic and no obvious vascular cavity like structure was formed. Conclusion: 1, the growth of the two cells in the direct co culture of MG63 and VECs in 1:1 was the most important. Well, cell growth, VECs nodular growth, MG63 growth in VECs nodules.2, IGF-1 expression value in MG63-VECs co culture system is superior to two individual culture; under the indirect co culture condition, the expression of IGF-1mRNA in co culture A group is significantly higher than that in co culture B group.3, through the tube type experiment of co culture system, it is proved to be bone fine. The vascular endothelial cells are more likely to form a tube type with the participation of the cells. The osteoblasts in the vascularization of bone tissue not only construct the basic structure of the bone tissue, but also as a type of tubular induced functional cells, and participate in the migration and formation of VECs.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R782

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 馬紅梅;鄒進(jìn);王躍中;艾紅軍;楊向紅;;人成骨細(xì)胞與臍靜脈血管內(nèi)皮細(xì)胞直接混合培養(yǎng)的實(shí)驗(yàn)研究[J];口腔醫(yī)學(xué);2007年06期

本文編號(hào)：2042030