本文選題：血管瘤 + 動(dòng)物模型　； 參考：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 血管瘤是嬰幼兒最常見的皮膚腫瘤,好發(fā)于頭面部和四肢等體表部位,是以血管內(nèi)皮細(xì)胞增生為主的良性腫瘤。其特點(diǎn)是在嬰幼兒出生后第1年迅速增長(zhǎng),在隨后的7-9年緩慢消退。一般將其分為增殖期、消退期和消退后期3個(gè)階段。其中相當(dāng)一部分血管瘤可以自然消退,不留下任何后遺癥;還有部分血管瘤可能破潰感染留下瘢痕,大約20%血管瘤會(huì)破壞正常組織甚至危及生命。但是其發(fā)病機(jī)理和消退機(jī)制到目前還不清楚。因此血管瘤的治療方法有很多,但是療效卻不確切。研究其發(fā)病機(jī)制不僅能夠預(yù)防血管瘤的發(fā)生,同時(shí)也有助于臨床的治療。 目前研究發(fā)現(xiàn),有助于血管生成的因子有:血管內(nèi)皮細(xì)胞生長(zhǎng)因子(VEGF)、雌二醇(oestradiol)、成纖維生長(zhǎng)因子(FGF)、血管生長(zhǎng)素(angiogenin)、轉(zhuǎn)化生長(zhǎng)因子(TGF)、α-腫瘤壞死因子(TNF-α)、血小板衍生生長(zhǎng)因子(PDGF)、白介素-8(IL-8)等。其中VEGF被認(rèn)為與血管瘤的生成、發(fā)展和消退有著重要的聯(lián)系。VEGF具有促進(jìn)血管化、維持內(nèi)皮細(xì)胞的活性、促進(jìn)內(nèi)皮細(xì)胞的遷移、促進(jìn)內(nèi)皮細(xì)胞的增殖、增加微血管通透性等特性。它是內(nèi)皮細(xì)胞(Endothelial cell,ECs)的特異性有絲分裂原,能選擇性的作用于EC,促進(jìn)其增殖、遷移,有利于血管生成。在腫瘤、缺血缺氧等情況下,VEGF及其受體呈高表達(dá)。而VEGF不足時(shí)會(huì)導(dǎo)致血管管腔閉合、血管退化。 目前利用基因干擾技術(shù)下調(diào)VEGF的表達(dá)在研究與治療腫瘤方面取得了極大的進(jìn)展。同時(shí)有學(xué)者發(fā)現(xiàn)增殖性血管瘤(嬰幼兒血管瘤,IH)不同于其它類型的血管畸形,其早期迅速的擴(kuò)張是血管內(nèi)皮細(xì)胞的非控性增生,這些增生是血管形成刺激因子或抑制因子水平異常引起的。其中VEGF被認(rèn)為在這種血管瘤的形成中起十分重要的作用。將VEGF作為一種新的治療靶點(diǎn),已部分應(yīng)用到血管瘤的臨床診斷和治療中。 本課題擬利用基因干擾技術(shù),通過體內(nèi)和體外實(shí)驗(yàn)研究,下調(diào)血管瘤動(dòng)物模型和嬰幼兒血管瘤血管內(nèi)皮細(xì)胞中VEGF基因的表達(dá),觀察血管瘤和血管瘤內(nèi)皮細(xì)胞的發(fā)展變化,同時(shí)探討血管瘤的增殖和消退機(jī)制。為進(jìn)一步臨床應(yīng)用研究奠定理論基礎(chǔ)。 1.裸鼠血管瘤移植模型的建立 目的:建立人毛細(xì)血管瘤裸鼠移植模型,探討血管瘤裸鼠模型建立的最佳條件。方法:將手術(shù)切除的雌激素受體陽性的兒童增生期血管瘤組織制成組織塊,植入20只裸鼠(BALB/c nude mice)皮下,每只4處,將20只裸鼠分為4個(gè)實(shí)驗(yàn)組。實(shí)驗(yàn)1組在移植后給予普通鼠食喂養(yǎng);實(shí)驗(yàn)2組在1組基礎(chǔ)上每周肌注雌二醇0.01 mg;實(shí)驗(yàn)3組在1組基礎(chǔ)上每周肌注雌二醇0.1 mg;實(shí)驗(yàn)4組在1組基礎(chǔ)上每周肌注雌二醇1mg,于移植后第30、60、90天切取移植瘤。移植瘤標(biāo)本進(jìn)行病理學(xué)光鏡檢查,用血管內(nèi)皮細(xì)胞單克隆抗體CD31、CD34、Ki-67行免疫組化染色。結(jié)果:移植后早期各組標(biāo)本內(nèi)皮細(xì)胞大量變性、壞死,30d后,單純喂養(yǎng)的實(shí)驗(yàn)1組及實(shí)驗(yàn)2組部分移植瘤開始吸收或形成膿腫及纖維化。實(shí)驗(yàn)3、4組移植瘤開始緩慢生長(zhǎng)。90d后實(shí)驗(yàn)1組實(shí)驗(yàn)2組移植瘤均未成活,實(shí)驗(yàn)4組移植瘤部分成活,而實(shí)驗(yàn)3組移植瘤全部成活。光鏡下成活的移植瘤與原血管瘤組織生物學(xué)特點(diǎn)相似。結(jié)論:不同劑量的雌激素對(duì)血管瘤裸鼠移植模型的建立有一定影響,適量的應(yīng)用雌激素可建立穩(wěn)定的人血管瘤裸鼠移植動(dòng)物模型。該模型可以應(yīng)用到基礎(chǔ)和臨床的血管瘤研究。 2.增殖期血管瘤內(nèi)皮細(xì)胞的培養(yǎng)和鑒定 目的:體外培養(yǎng)和鑒定血管瘤內(nèi)皮細(xì)胞(Hemangioma endothelial cell,HemEC)。方法:無菌條件下手術(shù)切取增生期血管瘤組織標(biāo)本,利用組織塊法進(jìn)行培養(yǎng)。用含20%胎牛血清的M199培養(yǎng)基,加入內(nèi)皮細(xì)胞生長(zhǎng)支持物(Endothelial cell growth supplement,ECGS,濃度為100μg/ml)在含5%CO2、37℃培養(yǎng)箱中培養(yǎng),每隔兩到三天換液,細(xì)胞鋪滿瓶底后傳代。用光學(xué)顯微鏡觀察細(xì)胞形態(tài)和生長(zhǎng)情況,并用免疫組織化學(xué)檢測(cè)細(xì)胞表達(dá)Ⅷ因子相關(guān)抗原和透射電鏡檢測(cè)鑒定細(xì)胞。結(jié)果:組織塊接種3天后有細(xì)胞開始從邊緣移出,細(xì)胞成多角形,1周左右混合成片,部分匯合成島狀。大約3周可以鋪滿瓶底,隨后消化傳代。傳代細(xì)胞成典型的“鵝卵石”樣排列鋪滿瓶底。細(xì)胞Ⅷ因子染色為陽性,透射電鏡顯示細(xì)胞質(zhì)內(nèi)含有ECs特征性的Weibel-Palade小體(W-P小體,呈點(diǎn)狀或卵形)。結(jié)論:利用組織塊法可以獲得純度較高的血管瘤內(nèi)皮細(xì)胞,該方法簡(jiǎn)單易用。 3.VEGF基因干擾質(zhì)粒對(duì)血管瘤內(nèi)皮細(xì)胞的影響 目的:應(yīng)用人血管內(nèi)皮生長(zhǎng)因子(VEGF)干擾質(zhì)粒,作用于體外培養(yǎng)的人增生期血管瘤內(nèi)皮細(xì)胞,觀察其對(duì)VEGF分泌及對(duì)血管瘤內(nèi)皮細(xì)胞的影響。方法:利用脂質(zhì)體法將真核表達(dá)質(zhì)粒pGCsi-U6NeoRFP-shVEGF轉(zhuǎn)染到HemECs。采用倒置熒光顯微鏡觀察質(zhì)粒轉(zhuǎn)染效果;用MTT法和酶聯(lián)免疫吸附(ELISA)法分析觀察shVEGF對(duì)HemECs的影響。結(jié)果:轉(zhuǎn)染組的HemECs與對(duì)照組相比, VEGF表達(dá)明顯減少,轉(zhuǎn)染組培養(yǎng)上清中的VEGF表達(dá)量在轉(zhuǎn)染的第1、3、5、7天分別為141.3±7.82、92.34±5.71、68.07±5.95及40.65±6.71pg/ml于其它組比較P0.05,且VEGF質(zhì)粒的轉(zhuǎn)染使VEGF的表達(dá)下降也促進(jìn)了細(xì)胞的凋亡。結(jié)論:轉(zhuǎn)染VEGF干擾質(zhì)粒可以明顯減少血管瘤內(nèi)皮細(xì)胞VEGF的表達(dá),同時(shí)也促進(jìn)了細(xì)胞的凋亡。 4.VEGF干擾慢病毒對(duì)裸鼠血管瘤移植模型的影響 目的:制作VEGF慢病毒干擾載體Lenti-siRNA-VEGF,將其作用于裸鼠血管瘤移植模型,觀察血管瘤的生長(zhǎng)和凋亡的變化,探討新的血管瘤防治方法。方法:將VEGF干擾質(zhì)粒包裝成慢病毒載體的Lenti-siRNA-VEGF。制作裸鼠血管瘤移植模型,將Lenti-siRNA-VEGF作用于移植模型,大體觀察瘤體變化,利用HE、免疫組化和TUNEL等方法觀察瘤體切片。再用Western-blot方法從蛋白質(zhì)水平探討VEGF被沉默后血管瘤的生長(zhǎng)和消退情況。結(jié)果:實(shí)驗(yàn)組瘤體在注射后1周瘤體體積開始變小,1個(gè)月后瘤體縮小變硬;對(duì)照組無明顯變化。HE檢測(cè)可見管腔閉塞,瘤體纖維化。CD31、CD34免疫組化檢測(cè)發(fā)現(xiàn)陽性細(xì)胞數(shù)分別為7.68±0.83、10.25±1.29(P0.05)。TUNEL檢測(cè)發(fā)現(xiàn)細(xì)胞凋亡較對(duì)照組明顯增多。Western-blot發(fā)現(xiàn)實(shí)驗(yàn)組VEGF蛋白表達(dá)明顯下降。結(jié)論:Lenti-siRNA-VEGF作用于移植模型可以明顯降低瘤體VEGF的表達(dá),促進(jìn)瘤體內(nèi)皮細(xì)胞凋亡。該方法為血管瘤進(jìn)一步實(shí)驗(yàn)和臨床研究提供一個(gè)新的方法。
[Abstract]:Hemangioma is the most common skin tumor in infants and infants. It is well distributed in the surface of the head, face and limbs. It is a benign tumor with vascular endothelial cell proliferation, which is characterized by a rapid growth of first years after birth and a slow decline in the next 7-9 years. It is generally divided into 3 stages of proliferation, regression and retreat. When a part of the hemangioma can naturally fade away without leaving any sequelae, some hemangiomas may break the infection and leave the scar, about 20% of the tumor will destroy the normal tissue or even life. But the mechanism and mechanism of the disease are still unclear. Therefore, there are many treatments for the hemangioma, but the curative effect is not accurate. Studying its pathogenesis can not only prevent the occurrence of hemangioma, but also help clinical treatment.
Current studies have found that the factors contributing to angiogenesis are vascular endothelial growth factor (VEGF), estradiol (oestradiol), fibroblast growth factor (FGF), angiogenin (angiogenin), transforming growth factor (TGF), alpha tumor necrosis factor (TNF- alpha), platelet derived growth factor (PDGF), and interleukins -8 (IL-8). VEGF is considered to be the same. The formation, development and regression of hemangioma have important connections with.VEGF, which can promote vascularization, maintain endothelial cell activity, promote endothelial cell migration, promote endothelial cell proliferation, and increase microvascular permeability. It is a specific mitogen for Endothelial cell (ECs), which can selectively act on EC and promote it. The proliferation and migration of the VEGF and its receptors are highly expressed in the cases of tumor, ischemia and hypoxia, and the insufficiency of VEGF will lead to the closure of the vascular lumen and the degeneration of blood vessels.
At present, the use of gene interference to reduce the expression of VEGF has made great progress in the research and treatment of tumor. At the same time, some scholars have found that proliferative hemangioma (infant hemangioma, IH) is different from other types of vascular malformation, its early rapid expansion is the non controlled proliferation of vascular endothelial cells, which are vascular formation spines. VEGF is considered to play an important role in the formation of this kind of hemangioma. VEGF, as a new therapeutic target, has been applied to the clinical diagnosis and treatment of hemangioma.
In this study, we use gene interference technique to reduce the expression of VEGF gene in the animal model of hemangioma and the vascular endothelial cells of infantile hemangioma through the study of in vivo and in vitro, to observe the development and changes of the endothelial cells of hemangioma and hemangioma, and to explore the mechanism of proliferation and regression of hemangioma, and lay the foundation for further clinical application. Theoretical basis.
Establishment of 1. model of hemangioma transplantation in nude mice
Objective: to establish a nude mouse model of human capillary hemangioma and to explore the optimum conditions for the establishment of a nude mouse model of hemangioma. Methods: the tissue block was made by the surgical resection of the oestrogen receptor positive hyperplastic hemangioma in children, and 20 nude mice (BALB/c nude mice) were implanted subcutaneously, each of the 20 nude mice was divided into 4 experimental groups. The 1 groups were moved to the experimental group. The 2 groups in the experimental group were injected estradiol 0.01 mg per week on the basis of 1 groups, and the 3 groups were injected estradiol 0.1 mg per week on the basis of the 1 groups, and the 4 groups were injected with estradiol 1mg per week on the basis of 1 groups. The transplanted tumor markers were examined by pathological light microscopy, and vascular endothelial cells were used for vascular endothelial cells. The monoclonal antibodies CD31, CD34, and Ki-67 were immunohistochemical staining. Results: after transplantation, the endothelial cells were denatured and necrotic at the early stage of the transplantation. After 30d, the 1 groups of experimental group and 2 groups of experimental groups began to absorb or form abscess and fibrosis. In the experimental 3,4 group, after the slow growth of.90d, the 1 groups of transplanted tumors were not transplanted. Survival, the experimental 4 groups of transplanted tumors survived, and all of the experimental 3 groups survived. The survival of the transplanted tumor was similar to that of the primary hemangioma. Conclusion: different doses of estrogen have a certain influence on the establishment of the transplanted model of hemangioma in nude mice. Proper use of estrogen can establish a stable transplantation of human hemangioma in nude mice. The model can be applied to basic and clinical studies of hemangioma.
Culture and identification of endothelial cells of 2. proliferative hemangioma
Objective: to cultivate and identify Hemangioma endothelial cell (HemEC) in vitro (HemEC). Methods: the tissue specimens of hyperplastic hemangioma were harvested under aseptic conditions and cultured with tissue block method. The M199 culture medium containing 20% fetal bovine serum was used to add Endothelial cell growth supplement, ECGS, and concentration. 100 g/ml) was cultured in a incubator containing 5%CO2,37. The cells were replaced every two to three days and the cells were spread through the bottom of the bottle. The cell morphology and growth were observed by optical microscopy. The cells expressed VIII related antigens and transmission electron microscopy were detected and identified by the immunohistochemical staining. The results were: the cells began to start from 3 days after inoculation. The edges are moved out and the cells are polygonal and mixed into slices for about 1 weeks. Some converts into an island shape. About 3 weeks can be filled with a bottle bottom and then digested. The passages are filled with a typical "cobblestone" arrangement. Cell VIII staining is positive. Transmission electron microscopy shows the cytoplasm of the cytoplasm containing ECs Weibel-Palade corpuscles (W-P corpuscles, Conclusion: tissue mass method can be used to obtain high purity hemangioma endothelial cells. This method is simple and easy to use.
Effect of 3.VEGF gene interference plasmid on hemangioma endothelial cells
Objective: To observe the effect of human vascular endothelial growth factor (VEGF) on the endothelial cells of human hyperplastic hemangioma in vitro, and to observe its effect on the secretion of VEGF and the endothelial cells of hemangioma. Method: transfection of eukaryotic expression plasmid pGCsi-U6NeoRFP-shVEGF to HemECs. by liposome method to observe plasmids by inverted fluorescence microscope. Transfection effect, the effect of shVEGF on HemECs was observed by MTT method and enzyme linked immunosorbent assay (ELISA). Results: the expression of VEGF in the transfected group decreased significantly compared with the control group, and the expression of VEGF in the transfected group was 141.3 + 7.82,92.34 + 5.71,68.07 + 5.95 and 40.65 + 6.71pg/ml in the other groups. Compared with P0.05, the transfection of VEGF plasmid reduced the expression of VEGF and promoted the apoptosis of the cells. Conclusion: transfection of VEGF interfering plasmids can obviously reduce the expression of VEGF in the endothelial cells of hemangioma, and also promote the apoptosis of the cells.
Effect of 4.VEGF interference Lentivirus on nude mice model of hemangioma transplantation
Objective: to make the VEGF lentivirus interference carrier, Lenti-siRNA-VEGF, to function in the model of hemangioma transplantation in nude mice, observe the changes of the growth and apoptosis of hemangioma, and discuss the new method of prevention and cure of hemangioma. Method: the VEGF interference plasmid is packaged into the Lenti-siRNA-VEGF. of the lentivirus vector to make the nude mouse hemangioma transplantation model, and Lenti-siRNA-VEGF The tumor body changes were observed and the tumor bodies were observed by HE, immunohistochemistry and TUNEL. The growth and decline of the tumor after the silence of VEGF were investigated by the Western-blot method. Results: the tumor volume began to become smaller after 1 weeks after the injection, and the tumor was narrowed and hardened after 1 months. There was no obvious change in the group with.HE detection. The occlusion of the lumen, the fibrotic.CD31 of the tumor, the number of positive cells detected by CD34 immunohistochemical detection were 7.68 + 0.83,10.25 + 1.29 (P0.05).TUNEL detection, respectively, and found that the apoptosis of the cells increased obviously than the control group.Western-blot found that the VEGF protein table of the experimental group decreased obviously. Conclusion: Lenti-siRNA-VEGF's effect on migration The implant model can significantly reduce the expression of VEGF and promote the apoptosis of the endothelial cells of the tumor. This method provides a new method for further experimental and clinical study of hemangioma.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 劉軍,宋建星,邢新,吳國祥,歐陽天祥,楊超,楊志勇;血管內(nèi)皮細(xì)胞生長(zhǎng)因子基因移植制備大鼠血管瘤模型[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2005年10期

2 虎小毅;楊壯群;宋勇;屠軍波;馬建明;徐泉;李鵬;;細(xì)胞懸液接種法血管瘤裸鼠動(dòng)物模型的建立[J];中國美容醫(yī)學(xué);2008年07期

3 馬戈甲;易成剛;孫智勇;裴蛟淼;王師平;夏煒;郭樹忠;楊力;;不同劑量雌激素對(duì)裸鼠血管瘤模型的影響[J];中國美容醫(yī)學(xué);2010年02期

4 徐殠,張志愿,陳萬濤,楊文君,王鑄鋼,王龍;轉(zhuǎn)基因小鼠血管瘤動(dòng)物模型建立[J];中華口腔醫(yī)學(xué)雜志;2003年05期

5 程立新,湯少明,羅少軍,郝新光;血管瘤組織中雌激素、雌激素受體表達(dá)及臨床意義[J];中華整形外科雜志;2003年01期

6 I-Shyan Sheen;Kuo-Shyang Jeng;Shou-Chuan Shih;Chih-Roa Kao;Wen-Hsing Chang;Horng-Yuan Wang;Tsang-En Wang;Li-Rung Shyung;Chih-Zen Chen;;Clinical significance of the expression of isoform 165 vascular endothelial growth factor mRNA in noncancerous liver remnants of patients with hepatocellular carcinoma[J];World Journal of Gastroenterology;2005年02期

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