不同细胞生长因子在皮瓣坏死创面愈合中作用和机制研究

发布时间：2018-06-18 10:37

  本文选题：大鼠 + 皮瓣　； 参考：《浙江大学》2014年博士论文

【摘要】：背景 皮瓣手术是整形重建和口腔颌面部手术中最重要的技术之一。然而,无论是在实验还是临床中,外科皮瓣坏死都是很难完全预防和避免的。皮瓣坏死往往导致皮瓣摘除。很多的研究都尝试通过药物,提高皮瓣成活率。然而,在这些研究中也有明显的一些不足。许多研究中没有动物模型解剖学证据；当坏死即将发生时,没有很好的解决方法；虽然一直对坏死的预防性研究很多,但关于促进坏死修复的研究很少,对皮瓣坏死创口治疗的研究更为鲜见。 KGF、EGF和VEGF是生长因子领域研究最多的几个生长因子,大量的文献都确定其分别特异性对上皮细胞、主要对上皮细胞、特异性对血管内皮细胞有很强的生长促进作用。KGF对上皮细胞的特异性作用,使其能促进上皮细胞增殖的同时,不促进纤维生长,不导致疤痕形成,成为促进上皮创口愈合最好的候选。EGF是临床上应用最多的生长因子之一,其对上皮的促进作用得到很多患者及医生的认同。VEGF是迄今为止发现的促进血管新生最有效的生长因子,能够极大地提高外科皮瓣的成活,预防皮瓣坏死。 皮瓣坏死的基因预防及治疗是一种极有潜力的方法,是未来研究的发展方向。当今皮瓣的基因研究主要是以生长因子作为治疗性基因完成的。如何以最佳的方法将外源性DNA导入皮瓣,使细胞接收并表达外源DNA,一直是值得大家研究和探讨的问题。 我们以腺病毒为载体(Ad),携带角化细胞生长因子(KGF)基因的方法,利用复制缺陷型腺病毒载体重组KGF基因,通过其强感染能力与转基因能力,在体外体内特异性表达有活性的KGF蛋白,发挥定向修复组织损伤的作用。另外我们直接在大鼠背部的坏死皮瓣区域中和皮肤中注入EGF和VEGF,观察联合应用生长因子是否会对皮瓣区坏死愈合起作用。 方法 1.Pac Ⅰ核酸内切酶酶切鉴定腺病毒质粒,测序检测是否质粒存在变异。Pac Ⅰ核酸内切酶酶切pAd-KGF,线性化后的pAd-KGF转染进HEK293(293)细胞后,制备Ad-KGF。应用293细胞扩增、纯化得到Ad-KGF病毒。应用于后续细胞及动物实验,并观察病毒转染293细胞后,细胞形态及病毒的感染能力。 2.Ad-KGF感染正常细胞系PA317,HUVEC,HaCaT,和293细胞后,观察病毒的感染的能力；提取受感染细胞的RNA,逆转录得到cDNA后,进行半定量PCR和荧光定量PCR,检测细胞内KGF的表达水平；通过ELISA,检测受感染细胞上清中KGF的表达水平；通过划痕实验模仿伤口,培养HaCaT细胞48小时,确定KGF对上皮细胞生长迁移的作用；将PA317细胞,HUVEC细胞和HaCaT细胞接种于Transwell小室,293细胞+Ad-KGF转染24小时(293+Ad-KGF),293细胞+Ad转染24小时(293+Ad),HaCaT,293细胞和293细胞+KGF(293+KGF)接种于下室内,观察KGF和Ad-KGF对成纤维细胞系,血管内皮细胞系和上皮细胞系的生长迁移作用。 3.我们首先解剖大鼠背部,研究确定大鼠背部血管走行。在传统大鼠背部皮瓣模型上,改良手术及实验过程,使皮瓣发生坏死。记录大鼠的体重,皮瓣坏死面积和愈合情况。在术后第15天,25天,和45天过量水合氯醛处死大鼠,取背部坏死皮瓣标本,进行组织化学染色分析。 4.在建立大鼠背部皮瓣模型之后,所有动物分为四组。将1ml PBS+dexamethasone(DXM),1ml PBS+Ad-KGF+DXM,or1ml PBS+Ad+DXM分别注入大鼠背部皮瓣坏死区皮下和创口边缘,分别于术后5天,10天,20天,和30天注射相同的药物。隔日记录体重,直至创面坏死愈合。术后第15天,25天,和35天过量水合氯醛处死大鼠,取背部坏死皮瓣标本,进行组织化学Masson染色、免疫组化染色分析和荧光定量PCR分析,上皮及上皮下组织的生长情况,上皮的生长厚度,取血清进行ELISA检测KGF表达水平。 5.首先通过细胞迁移实验确定EGF和VEGF对PA317,HUVEC,和HaCaT细胞迁移的活性。建立大鼠背部皮瓣模型,坏死发生后,沿创口边缘及坏死皮下注入1ml PBS+EGF (150mg/kg),1ml PBS+VEGF (10μg), or1ml PBS+EGF (150μg/kg)+VEGF(10μg)。隔日记录体重并注射相同的药物,直至创面坏死愈合。术后第15天和25天过量水合氯醛处死大鼠,取背部坏死皮瓣标本,进行组织化学Masson染色和免疫组化染色分析创缘上皮及上皮下组织的生长情况,上皮的生长厚度,并取血清进行ELISA检测。 结果 1.酶切鉴定结果与设计和文献相符,证明载体pAd-KGF构建正确。pAd-KGF质粒测序结果显示起始位点：第185bp,ATG。终结位点：第679bp,TAA。全序列对比小鼠KGF基因序列,二者完全匹配,不存在突变。病毒转染正常293细胞24小时,开始出现细胞变圆；荧光显微镜镜下观察,部分293细胞开始表达大量荧光蛋白。转染第96小时后,所有293产生细胞病理效应,显微镜下观察所有细胞呈圆球形葡萄样改变,漂浮于培养液中,荧光显微镜镜下观察293细胞呈圆球形,表达大量荧光蛋白。重组腺病毒Ad-KGF和Ad在293细胞内大量扩增,并收集后。测得Ad-KGF病毒的效价滴度为2×1011PFU/ml,Ad病毒的效价滴度为3×1012PFU/ml。 2.pAd-KGF为构建腺病毒质粒。半定量PCR证明pAd-KG内存在KGF表达基因。经过基因重组后的腺病毒Ad-KGF具有感染正常细胞,于正常细胞内表达KGF的能力。正常的293细胞不能表达KGF的mRNA,PA317细胞能分泌KGF,表达KGF mRNA。Ad-KGF感染293细胞后,细胞成功表达KGF mRNA,而在293细胞和293+空病毒Ad内不能表达KGF mRNA。通过ELISA检测培养液上清中外分泌的KGF含量。检测证明Ad空病毒转染细胞后48小时,上清液中不分泌KGF蛋白,而Ad-KGF转染细胞的后的48小时,细胞培养上清中能够检测到大量的KGF表达,且浓度随着Ad-KGF的滴度增加而增加。通过划痕实验,证明在KGF作用下,HaCaT上皮细胞的迁移能力显著增加。而对照组细胞开始出现凋亡现象。Transwell细胞迁移实验证明KGF能有效促进HaCaT上皮细胞生长迁移,而对HUVEC血管内皮细胞作用不明显,对PA317成纤维细胞没有明显作用。 3.大鼠背部七条静脉和一侧有超过二十条动脉分布。手术后三天,大鼠的体重会稍有下降,然后稳定上升。PBS组和空白组中坏死面积在9天内达到最高峰。从9至47天中,坏死面积逐渐减少,在47天坏死几乎完全愈合。组织病理检查中,可见术后15天,坏死创口边缘上皮细胞增生明显,有少量纤维成分生成,创口愈合区有大量小脉管形成和大量不成熟的纤维成分形成。至第35天,上皮增生修复较前明显弱,但较正常上皮,厚度仍明显增大。 4.成功建立大鼠背部皮瓣坏死模型后,四组动物术后体重均有轻度下降,从第三天开始,体重逐步上升,其中以DXM组的体重上升最明显。术后第五天,坏死成活区域分界清晰。术后第十天,坏死面积达到最大。随着不同药物的治疗,坏死面积在各组间都明显下降,且Ad-KGF组坏死面积缩小最为明显。Masson染色显示术后15天,四组坏死创口边缘上皮都明显增生,其中Ad-KGF组上皮细胞增生最多。术后35天,Ad-KGF组坏死伤口已经愈合,而其它三组还有较大的坏死面积。术后15天和25天,上皮细胞厚度在Ad-KGF组增生最为明显,术后35天上皮厚度与其它组间没有大的差异。 免疫组织化学分析显示,只有在Ad-KGF组的上皮和间充质细胞中有强阳性的KGF表达,而其它三组均为阴性。Anti-CD34免疫组织化学染色显示四组的小血管生成数目相当 5.PA317在EGF, VEGF, VEGF+EGF,或PA317作用下,没有显著性的生长差异。HUVEC对VEGF表现出明显生长加速,在EGF和VEGF联合作用下,生长更加明显,而与PA317共培养时,也有少量生长改变。HaCaT细胞在EGF和VEGF+EGF联合作用下都是表现出生长迁移能力提高。建立动物模型后,分组如下:PBS, EGF, VEGF, EGF+VEGF。四组动物术后体重均有轻度下降,从第三天开始,体重逐步上升,其中以EGF组的体重上升最少。随着不同药物的治疗,坏死面积在各组间都明显下降,且VEGF+EGF组坏死面积缩小最为明显。 术后15天和25天,Anti-CD34免疫组织化学染色显示EGF,VEGF和VEGF+EGF组的小微血管生成数目相当,都显著高于PBS组。 结论 1. pAd-KGF经过酶切鉴定和测序鉴定具有KGF完全匹配的基因组。成功构建Ad.KGF和Ad,且具有感染细胞的能力。Ad.KGF和Ad经过扩增纯化的滴度为2×1011PFU/ml和3×1012PFU/ml,可以满足细胞及大鼠皮瓣实验的要求。 2. Ad-KGF和Ad可以高效转染PA317、HUVEC和HaCaT细胞,并在转染后高效表达活性KGF、KGF可有效促进HaCaT细胞生长,而对PA317、HUVEC细胞作用不明显。 3.本实验在确定大鼠背部血供系统基础上,设计形成一套简便且易于推广的手术和皮瓣坏死模型流程,成功建立了稳定的大鼠背部皮瓣坏死伤口模型,可以进行皮瓣坏死为目的基因或药物实验研究。 4.使用高表达KGF的腺病毒重组局部转染大鼠背部皮瓣坏死伤口,能有效促进坏死伤口的愈合。 5. EGF-VEGF联合应用能促进改良大鼠背部坏死创面愈合。
[Abstract]:background
Flap surgery is one of the most important techniques in plastic reconstruction and oral and maxillofacial surgery. However, the necrosis of the skin flap is difficult to prevent and avoid in both experimental and clinical trials. Skin flap necrosis often leads to the removal of the skin flap. Many studies have tried to improve the survival rate of the skin flap. There are some obvious shortcomings. There is no evidence of animal model anatomy in many studies; there is no good solution to the necrosis when the necrosis is about to occur; although there are many preventive studies on necrosis, few studies have been made on the promotion of necrosis, and the research on the treatment of skin flap necrosis is more rare.
KGF, EGF and VEGF are the most important growth factors in the field of growth factor. A large number of literatures have identified the specificity of the epithelial cells, mainly to the epithelial cells, and the specific effects of the specific growth of the vascular endothelial cells on the specific effect of.KGF on the epithelial cells, so that they can promote the proliferation of epithelial cells and do not promote the proliferation of epithelial cells. Fiber growth, no scar formation, the best candidate.EGF to promote the healing of the epithelial wound is one of the most clinically applied growth factors. The promotion of the epithelium has been recognized by many patients and doctors..VEGF is the most effective growth factor that has been discovered so far to promote angiogenesis and can greatly improve the surgical flap. The survival of the skin to prevent the necrosis of the skin flap.
The gene prevention and treatment of skin flap necrosis is a potential method, which is the development direction of future research. The research on the gene of skin flap is mainly done by the growth factor as the therapeutic gene. How to introduce exogenous DNA into the skin flap in the best way and make the cells to receive and express foreign DNA has always been worth studying and exploring. It's a question.
We use the adenovirus vector (Ad) and the method of carrying keratinocyte growth factor (KGF) gene to restructure the KGF gene with the replication defective adenovirus vector, and express the active KGF protein in the body by its strong infection ability and transgene ability in vitro, and play the role of directed repair tissue damage. In addition, we directly in the rat EGF and VEGF were injected into the necrotic flap area of the back and the skin to observe whether the combined growth factor could play a role in the necrosis of the flap area.
Method
1.Pac I endonuclease was used to identify adenovirus plasmids, sequencing and detecting whether plasmids were mutant.Pac I endonuclease pAd-KGF, pAd-KGF transfected into HEK293 (293) cells after linearized pAd-KGF were transfected into HEK293 (293) cells, and Ad-KGF. was amplified and purified to get Ad-KGF virus. It was applied to subsequent cell and animal experiments, and the virus transfection 293 was observed. Cells, cell morphology and virus infection.
2.Ad-KGF infected normal cell lines PA317, HUVEC, HaCaT, and 293 cells, observed the ability of the virus infection, extracted the RNA of the infected cells, and then reverse transcriptase cDNA to carry out the semi quantitative PCR and fluorescent quantitative PCR to detect the expression level of KGF in the cells; detect the KGF expression level in the infected cell supernatant by ELISA; through scratching the scar. HaCaT cells were cultured for 48 hours to determine the effect of KGF on the growth and migration of epithelial cells. PA317 cells, HUVEC cells and HaCaT cells were inoculated in the Transwell chamber, the 293 cells were transfected with +Ad-KGF for 24 hours (293+Ad-KGF), and 293 cells were transfected +Ad (293+Ad), HaCaT, 293 and 293 cells were inoculated in the lower room. The growth and migration effects of GF and Ad-KGF on fibroblasts, vascular endothelial cells and epithelial cell lines.
3. we first dissected the back of the rat and studied the back blood vessels of the rat. On the traditional rat back flap model, we improved the operation and the experimental process to make the skin flap necrotic. The weight of the rats, the necrotic area and the healing of the skin flap were recorded. The rats were killed at the fifteenth day, 25 days, and 45 days after the operation, and the necrotic skin flap of the back was taken. Specimens were analyzed by histochemical staining.
4. after the model of the rat back flap was established, all the animals were divided into four groups. 1ml PBS+dexamethasone (DXM), 1ml PBS+Ad-KGF+DXM and or1ml PBS+Ad+DXM were injected into the necrotic region of the dorsal skin flap of the rat back and the edge of the wound respectively. The same drugs were injected at 5 days, 10 days, 20 days, and 30 days after the operation respectively. The rats were killed at fifteenth days, 25 days, and 35 days with chloral hydrate, and the specimens of the back necrotic skin flap were taken to perform histochemical Masson staining, immunohistochemical staining and fluorescence quantitative PCR analysis, the growth of epithelium and subcutaneous tissue, the thickness of epithelial growth, and the serum level of ELISA to detect the expression of KGF.
5. first to determine the activity of EGF and VEGF on the migration of PA317, HUVEC, and HaCaT cells through cell migration experiments. The rat model of the back flap was established. After the necrosis occurred, 1ml PBS+EGF (150mg/kg) was injected along the edge of the wound and necrotic subcutaneously, 1ml PBS+VEGF (10 mu g), or1ml (150 mu) (10 mu). In the fifteenth and 25 days after the operation, the rats were killed with chloral hydrate, and the necrotic skin flap of the back was taken. The tissue chemical Masson staining and immunohistochemical staining were used to analyze the growth of the epithelial and subcutaneous tissue, the thickness of the epithelial growth, and the ELISA test.
Result
The results of the 1. enzyme digestion were consistent with the design and the literature. The results showed that the vector pAd-KGF constructed correct.PAd-KGF plasmid sequencing results showed the starting site: 185bp, ATG. terminating site: the whole sequence of 679bp and TAA. was compared with the KGF gene sequence of mice, the two were completely matched, without mutation. The virus transferred to normal 293 cells for 24 hours and began to appear cell circle. Under the fluorescence microscope, a large number of 293 cells began to express a large number of fluorescent proteins. After ninety-sixth hours transfection, all 293 of the 293 cells had the pathological effects. Under the microscope, all the cells were observed in the spheroidal grapevine like changes, floating in the culture medium. Under the fluorescence microscope, the 293 cells were round and expressed a large number of fluorescent proteins. Recombinant adenovirus A was expressed. D-KGF and Ad were amplified and collected in 293 cells. The titer of Ad-KGF virus was 2 * 1011PFU/ml and the titer of Ad virus was 3 * 1012PFU/ml..
2.pAd-KGF is the construction of adenovirus plasmid. Semi quantitative PCR shows that pAd-KG is in KGF expression gene. After recombinant adenovirus Ad-KGF has the ability to infect normal cells and express KGF in normal cells. Normal 293 cells can not express mRNA of KGF, PA317 cells can secrete KGF and express KGF mRNA.Ad-KGF to infect 293 cells. The work expressed KGF mRNA, but the content of KGF secreted in the supernatant of KGF mRNA. was detected in the 293 cells and 293+ empty virus Ad by ELISA. The detection showed that 48 hours after the transfection of the Ad empty virus, the KGF protein was not secreted in the supernatant, and a large amount of KGF was detected in the cell culture supernatant for 48 hours after the Ad-KGF transfection of the cells. The expression and concentration increased with the increase of the titer of Ad-KGF. Through the scratch test, it was proved that the migration ability of HaCaT epithelial cells increased significantly under the action of KGF, while the apoptosis in the control group began to occur in the.Transwell cell migration experiment, which proved that KGF could effectively promote the migration of HaCaT epithelial cells, and the effect on HUVEC vascular endothelial cells. It is not obvious, and has no obvious effect on PA317 fibroblasts.
There were more than twenty arteries on the seven veins and side of the back of the 3. rats. On the three day after the operation, the body weight of the rats decreased slightly, then the necrotic area in the.PBS group and the blank group reached the peak in 9 days. From 9 to 47 days, the necrotic area gradually decreased and the necrosis was almost completely healed at 47 days. The histopathological examination showed postoperative surgery. On the 15 day, the epithelial cells of the necrotic wound edge proliferated obviously, with a small amount of fibrous composition, and a large number of small vessels formed and a large number of immature fibers formed in the wound healing area. To the thirty-fifth day, the epithelia repair was significantly weaker than before, but the thickness of the epithelial cells was significantly increased.
4. after the rat model of the back flap necrosis was successfully established, the weight of the four groups decreased slightly. From the third day, the body weight increased gradually. The body weight of the DXM group increased most obviously. The necrotic area was clearly defined at fifth days after the operation. The worst dead surface was the largest after tenth days of operation. With the treatment of different drugs, the necrotic area was found. At 15 days after the operation, the four groups of necrotic wound epithelia were obviously proliferated, and the epithelial cells of group Ad-KGF were most proliferated. The necrosis wound in group Ad-KGF was healed at 35 days after operation, and the other three groups had larger necrotic area. 15 and 25 days postoperatively, 15 and 25 days after the operation. The thickness of skin cells in Ad-KGF group was the most obvious. There was no significant difference in epithelial thickness between 35 days after operation and those of other groups.
Immunohistochemical analysis showed that there was a strong positive KGF expression in the epithelial and mesenchymal cells of the Ad-KGF group, while the other three groups were negative.Anti-CD34 immunohistochemical staining, which showed that the number of small blood vessels in the four groups was equal.
Under the action of EGF, VEGF, VEGF+EGF, or PA317, there was no significant growth difference between.HUVEC and VEGF, which showed obvious growth acceleration. Under the combined action of EGF and VEGF, the growth of 5.PA317 was more obvious. While co culture with PA317, a small amount of growth and change of.HaCaT cells showed growth and migration ability. After the animal model was established, the groups were grouped as follows: PBS, EGF, VEGF, EGF+VEGF. four groups had a slight decrease in weight after operation. From the third day, the body weight increased gradually, and the body weight of the EGF group increased least. With the treatment of different drugs, the necrotic area decreased significantly in each group, and the necrotic area in the VEGF+EGF group narrowed the most obvious.
On the 15 and 25 days after operation, Anti-CD34 immunohistochemical staining showed that the number of small microvessels in EGF, VEGF and VEGF+EGF groups was similar, which were significantly higher than those in PBS group.
conclusion
1. pAd-KGF was identified and sequenced by enzyme digestion and sequencing. The genome was fully matched with KGF. The successful construction of Ad.KGF and Ad, and the ability of.Ad.KGF and Ad to infect cells, was 2 * 1011PFU/ml and 3 x 1012PFU/ml, which could meet the requirements of cell and rat skin flap experiments.
2. Ad-KGF and Ad can efficiently transfect PA317, HUVEC and HaCaT cells, and express the active KGF after transfection. KGF can effectively promote the growth of HaCaT cells, but the effect on PA317 and HUVEC cells is not obvious.
3. on the basis of determining the blood supply system on the back of the rat, a simple and easy to popularize operation and the flow pattern of the skin flap necrosis was designed. A stable model of the necrotic wound of the dorsal flap of the rat was successfully established, and the necrosis of the skin flap could be used as the target gene or the drug experiment.
4. recombinant adenovirus with high expression of KGF can be used to transfect the dorsal skin flap of rats to necrotic wound effectively, which can effectively promote the healing of necrotic wounds.
5. EGF-VEGF combined application can promote the healing of necrotic wounds on the back of rats.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R782.2
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本文编号：2035191