不同剂量重组人生长激素皮下注射对大鼠背部随意皮瓣存活影响的实验研究
发布时间：2019-06-17 09:13
【摘要】：研究背景:随着对创面修复的不断深入研究,越来越多的目光转向皮瓣修复。这种修复方法不仅能完成对创面的完全覆盖,还能解决创基薄弱、骨及肌腱外露等传统手术修复困难的难治性创面,因此,各大外科对皮瓣修复的应用也日益频繁。而随意皮瓣作为其中最常用方法之一,其成活率的提高一直广受医生、学者关注和研究。随意皮瓣内缺少知名血管及缺血-再灌注损伤(I-R损伤)仍极大的限制着随意皮瓣的成活。近年来,随着皮瓣设计的不断完善,比如:最佳长宽比例、皮瓣形状、术中不同血管吻合方式、延迟行皮瓣移植、加强创面护理、术后定期高压氧治疗以及更换敷料时各种生长因子的应用等方法的大量推行,皮瓣成活率显著上升,但临床应用中皮瓣坏死的情况仍屡见不鲜。如何能最大程度避免皮瓣中I-R损伤的发生,提高其皮瓣生存率成为近年来的热门研究方向。重组人生长激素(rhGH)在以往研究中已被证实了有增强血浆蛋白mRNA表达和促进血清白蛋白(ALB)合成、提高血糖、分解脂质、免疫调控、增生血管、减少蛋白分解,减少氮丢失,促进伤口愈合的作用。在此基础之上,rhGH的临床应用不断增加,并扩展到烧伤创面及皮瓣存活方面。皮瓣移植后补充rhGH可以明显降低炎症因子白细胞介素-1(IL-1)、白细胞介素-6(IL-6)、肿瘤坏死因子-a(TNF-a)的表达,减少中性粒细胞的聚集。并刺激单核细胞的迁移、促进超氧阴离子生成,使机体的抵抗力得以提高,从而减轻炎症反应。彭丽霞等研究表明,大鼠皮下注射rhGH后可促进皮瓣中血管内皮生长因子(VEGF)的表达,从而起到促进血管新生的作用,使皮瓣坏死率降低。但其中剂量的选择尚未进行明确实验证明。基于上述原因,本次实验以大鼠为模型,验证不同剂量rhGH对大鼠背部随意皮瓣的影响,以了解其皮下注射的有效剂量。目的:通过皮下注射rhGH影响大鼠背部随意皮瓣成活,并采用不同注射剂量,观察不同剂量的rhGH对大鼠背部随意皮瓣影响程度,找出最佳注射剂量,为其临床应用前景做出初步评估。方法:健康成年SD大鼠64只,均在其背部正中设计蒂在尾端的大小为2cm*4cm的随意皮瓣,并采用随机分配原则,将大鼠分为A(生理盐水组)、B(0.05IU/Kg rhGH组)、C(0.1 IU/Kg rhGH组)、D(0.2 IU/Kg rhGH组)四组。术后即刻开始皮下注射药物,连续7天,一日一次。四组均于术后第7天拍照计算皮瓣成活率,并取所有皮瓣远端进行实验结果观察。将标本进行HE染色、免疫组化染色观察VEGF及CD34在组织中的表达情况,计算皮瓣微血管密度(MVD),数据进行统计学分析,明确实验目的。结果:1、皮瓣大体观察:A组皮瓣近端皮瓣成活区呈粉红色,毛发稀疏。中段及远端部分坏死,呈黑色,质硬,弹性明显降低,无毛发生长,针刺不出血,皮瓣下淡红色分泌物较多。B组皮瓣近端色泽红润,成活尚可,中段开始逐渐色泽晦暗,呈棕色,少量黑色痂壳附着,弹性降低,针刺可见少量出血,毛发逐渐稀疏,皮瓣下存在少量分泌物,直至远端色棕黑。C组及D组皮瓣成活情况相似,大部分皮瓣存活良好,近端及中段呈淡红色,弹性与正常组织相似,无明显红肿等情况。远端及少许边缘部位,可见部分棕黑色坏死区,面积远小于A、B组,针刺出血,皮瓣下无明显积血积液。2、皮瓣成活率:经实验及统计学分析可以得出:A组(对照组)为(57.72±2.03)%,B组为(72.75±0.97)%,C组为(81.37±1.49)%,D组为(82.31±1.59)%。将全部数据进行单因素方差分析(one-way ANOVA)得出:P0.05,各组数据总体均数不全相等。再运用LSD法将各组数据进行两两比较可得:各实验组与对照组进行比较,PO.05,差异有统计学意义,即rhGH皮下注射可提高皮瓣成活率。B组与C、D两组比较结果可见:PO.05,差异有统计学意义,可得出低剂量组rhGH对皮瓣成活率的促进作用低于中、高剂量组;C、D组两组统计学比较得出PO.05,差异无统计学意义,故中、高剂量rhGH促进皮瓣成活无显著性差异。3、HE染色可见:A组镜下见组织结构混乱不清,各层结构显示不明,组织明显水肿,大量中性粒细胞及单核细胞浸润,呈急性炎症反应改变,少量薄壁血管生长,无明显管状血管,部分纤维组织生长。B组真皮下组织较混乱,组织水肿及中性粒细胞及单核细胞浸润较A组减轻,大量毛细血管生长,肉芽清晰可见。C组及D组镜下少许炎症细胞浸润,组织结构显示清晰,新生管状血管生成,内可见红细胞充盈。4、CD34及VEGF在组织中的表达:运用免疫组化染色观察组织内CD34及VEGF的表达强弱,结果明确可见CD34染色玻片中CD34呈棕黄色颗粒,主要分布于细胞浆毛细血管周围,在B、C、D组中的表达明显高于A组,D组最高,C组其次。而VEGF免疫组化检测中呈药物干预组可见大量棕黄色颗粒分布成团状,对照组则呈点状散在分布,分布密度D组≥C组B组A组。5、微血管密度检测(MVD):将CD34免疫组化染色玻片置于100倍光镜视野下,每张玻片选取6个视野计数微血管数目并取其均值。A组:(5.00±1.46)条/mm2,B组(10.55±1.78)条/mm2,C组(14.40±1.63)条/mm2,D组(15.19±1.46)条/mm2,进行单因素方差分析后得出,P0.05,各组总体均数不完全相等,即表示各组间差异有统计学意义。再LSD法两两比较得出,药物干预组与对照组P0.05,有显著差异,C、D组与B组比较得出P0.05,低剂量组微血管密度偏低。C、D两组比较P0.05,差异无统计学意义。结论:1)重组人生长激素皮下注射可促进大鼠背部随意皮瓣内CD34及VEGF的表达增加,从而促进皮瓣内新生血管生成;2)重组人生长激素能提高大鼠随意皮瓣的成活率;3)0.1-0.2IU/kg剂量的重组人生长激素对皮瓣的作用最强,可作为最佳药物剂量使用。
[Abstract]:Background: With the development of wound repair, more and more eyes have been turned to skin flap repair. The method not only can complete the complete coverage of the wound surface, but also can solve the refractory wound surface which is difficult to repair the traditional operation such as the weakness of the wound, the bone and the exposure of the tendon and the like, so the application of the major surgery to the skin flap repair is also more frequent. As one of the most commonly used methods, the survival rate of the free skin flap has been widely accepted by the doctors, scholars and scholars. The lack of well-known vascular and ischemia-reperfusion injury (I-R injury) in the free flap still greatly limits the survival of the free flap. In recent years, with the continuous improvement of the design of the skin flap, for example, the optimal aspect ratio, the shape of the skin flap, the different vascular anastomosis methods in the operation, the delayed-row skin flap transplantation, the strengthening of the wound care, The high-pressure hyperbaric oxygen treatment and the application of various growth factors in the replacement of dressing, the survival rate of the skin flap increased significantly, but the case of the flap necrosis in clinical application is still common. How to minimize the occurrence of I-R injury in the skin flap and to improve the survival rate of the skin flap is the hot research direction in recent years. The recombinant human growth hormone (rhGH) has been proved to be a function of enhancing the expression of plasma protein mRNA and promoting the synthesis of serum albumin (ALB), improving blood sugar, decomposing lipid, regulating immunity, proliferating blood vessels, reducing protein decomposition, reducing nitrogen loss and promoting wound healing. On this basis, the clinical application of rhGH is increasing, and it is extended to the burn wound and the survival of the skin flap. RhGH can significantly reduce the expression of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a) and reduce the aggregation of neutrophils. And the migration of the mononuclear cells is stimulated, the generation of the superoxide anion is promoted, the resistance of the body is improved, and the inflammatory reaction is reduced. The results show that the expression of vascular endothelial growth factor (VEGF) in the skin flap can be promoted by subcutaneous injection of rhGH in the rat, so as to play a role in promoting the angiogenesis, and the necrosis rate of the skin flap is reduced. However, the selection of the dose has not been clearly demonstrated. Based on the above-mentioned reasons, the effect of rhGH on the free skin flap on the back of the rat was verified by using the rat model, so as to know the effective dose of the subcutaneous injection. Objective: To study the effect of rhGH on the random skin flap of the back of the rat by subcutaneous injection of rhGH, and to observe the effect of rhGH on the random skin flap of the back of the rat, to find out the optimal injection quantity and to make a preliminary assessment of its clinical application prospect. Methods:64 healthy adult SD rats were randomly divided into four groups: A (normal saline group), B (0.05 IU/ Kg rhGH group), C (0.1 IU/ Kg rhGH group) and D (0.2 IU/ Kg rhGH group). The subcutaneous injection of the drug was started immediately after the operation for 7 consecutive days and one time a day. The survival rate of the flap was calculated on the 7th day after the operation, and the distal end of all the flaps was taken for observation. The expression of VEGF and CD34 in the tissue was observed by HE staining and immunohistochemical staining. The microvessel density (MVD) and data of the skin flap were calculated. Results:1. The flap of the flap in group A was found to be pink and the hair was sparse. The middle section and the distal part were necrotic, black, hard, elastic, no hair growth, no bleeding, and more light red secretion under the skin flap. The color of the proximal end of the group B skin flap was ruddy, the survival was fair, the middle section was gradually dark, brown, a small amount of black skin attached to the skin, the elasticity decreased, the needle stick can be seen with a small amount of bleeding, the hair is gradually sparse, and a small amount of secretion is present under the skin flap until the distal color is dark brown. The survival of the skin flaps of group C and D was similar, most of the flaps survived well, the proximal and middle sections were light red, the elasticity was similar to that of the normal tissues, and there was no obvious swelling and swelling. The area of the distal and a few marginal areas, visible part of the brown-black necrotic area, was much smaller than that of group A, group B, acupuncture and hemorrhage, and no apparent accumulation of blood in the skin flap.2. The survival rate of the skin flap: The experimental and statistical analysis showed that the group A (control group) was (57.72-2.03)%. Group B (72.75-0.97)%, group C (81.37-1.49)%, group D (82.31-1.59)%. One-way ANOVA for all data was obtained: P0.05, the total number of data in each group was not completely equal. The data of each group were obtained by means of the LSD method: the experimental group and the control group were compared, and the difference was statistically significant, that is, the survival rate of the skin flap could be increased by subcutaneous injection of rhGH. The results of the comparison between group B and C and D were found in the two groups: PO.05, the difference was statistically significant, and the effect of rhGH on the survival rate of the skin flap was lower than that of the middle and high dose groups. The results showed that there was no significant difference in the survival of the skin flap with high dose of rhGH. No significant tubular vessel, partial fibrous tissue growth. The tissue edema and the infiltration of neutrophils and monocytes in group B were lower than that in group A, and a large number of capillary growth and granulation were clearly visible. The expression of CD34 and VEGF in the tissue was observed by immunohistochemical staining. The results showed that the CD34 in the CD34 stained slide showed brownish yellow particles, which were mainly distributed around the blood capillary of the cytoplasm, and the expression in the group B, C and D was higher than that in group A, the highest in group D and the second in group C. In the expression of VEGF, a large number of brown yellow particles were found in the drug-intervention group, and the control group was in the form of dot-like powder, and the control group was distributed in group B, Group A, Group B, Group B, and the density of microvessel density was detected (MVD): the CD34 immunohistochemical staining slide was placed in the field of 100-fold light microscope. The number of microvessels in 6 fields of view was selected for each slide and the mean value was taken. Group A: (5.00-1.46)/ mm2, group B (10.55-1.78)/ mm2, group C (14.40-1.63)/ mm2, group D (15.19-1.46)/ mm2, and after single-factor analysis of variance, the overall average number of the groups was not exactly equal, that is, the difference between groups was statistically significant. The results showed that there was a significant difference between the drug intervention group and the control group, and the microvessel density of the low-dose group was lower than that of the control group (P0.05). There was no significant difference between the two groups (P0.05). Conclusion:1) The injection of recombinant human growth hormone can promote the expression of CD34 and VEGF in the free flap of the back of the rat, thereby promoting the generation of the new blood vessel in the skin flap;2) the recombinant human growth hormone can improve the survival rate of the free skin flap of the rat; 3) The effect of the recombinant human growth hormone of 0.1-0.2 IU/ kg on the skin flap is the strongest, and can be used as the optimal drug dosage.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622
[Abstract]:Background: With the development of wound repair, more and more eyes have been turned to skin flap repair. The method not only can complete the complete coverage of the wound surface, but also can solve the refractory wound surface which is difficult to repair the traditional operation such as the weakness of the wound, the bone and the exposure of the tendon and the like, so the application of the major surgery to the skin flap repair is also more frequent. As one of the most commonly used methods, the survival rate of the free skin flap has been widely accepted by the doctors, scholars and scholars. The lack of well-known vascular and ischemia-reperfusion injury (I-R injury) in the free flap still greatly limits the survival of the free flap. In recent years, with the continuous improvement of the design of the skin flap, for example, the optimal aspect ratio, the shape of the skin flap, the different vascular anastomosis methods in the operation, the delayed-row skin flap transplantation, the strengthening of the wound care, The high-pressure hyperbaric oxygen treatment and the application of various growth factors in the replacement of dressing, the survival rate of the skin flap increased significantly, but the case of the flap necrosis in clinical application is still common. How to minimize the occurrence of I-R injury in the skin flap and to improve the survival rate of the skin flap is the hot research direction in recent years. The recombinant human growth hormone (rhGH) has been proved to be a function of enhancing the expression of plasma protein mRNA and promoting the synthesis of serum albumin (ALB), improving blood sugar, decomposing lipid, regulating immunity, proliferating blood vessels, reducing protein decomposition, reducing nitrogen loss and promoting wound healing. On this basis, the clinical application of rhGH is increasing, and it is extended to the burn wound and the survival of the skin flap. RhGH can significantly reduce the expression of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a) and reduce the aggregation of neutrophils. And the migration of the mononuclear cells is stimulated, the generation of the superoxide anion is promoted, the resistance of the body is improved, and the inflammatory reaction is reduced. The results show that the expression of vascular endothelial growth factor (VEGF) in the skin flap can be promoted by subcutaneous injection of rhGH in the rat, so as to play a role in promoting the angiogenesis, and the necrosis rate of the skin flap is reduced. However, the selection of the dose has not been clearly demonstrated. Based on the above-mentioned reasons, the effect of rhGH on the free skin flap on the back of the rat was verified by using the rat model, so as to know the effective dose of the subcutaneous injection. Objective: To study the effect of rhGH on the random skin flap of the back of the rat by subcutaneous injection of rhGH, and to observe the effect of rhGH on the random skin flap of the back of the rat, to find out the optimal injection quantity and to make a preliminary assessment of its clinical application prospect. Methods:64 healthy adult SD rats were randomly divided into four groups: A (normal saline group), B (0.05 IU/ Kg rhGH group), C (0.1 IU/ Kg rhGH group) and D (0.2 IU/ Kg rhGH group). The subcutaneous injection of the drug was started immediately after the operation for 7 consecutive days and one time a day. The survival rate of the flap was calculated on the 7th day after the operation, and the distal end of all the flaps was taken for observation. The expression of VEGF and CD34 in the tissue was observed by HE staining and immunohistochemical staining. The microvessel density (MVD) and data of the skin flap were calculated. Results:1. The flap of the flap in group A was found to be pink and the hair was sparse. The middle section and the distal part were necrotic, black, hard, elastic, no hair growth, no bleeding, and more light red secretion under the skin flap. The color of the proximal end of the group B skin flap was ruddy, the survival was fair, the middle section was gradually dark, brown, a small amount of black skin attached to the skin, the elasticity decreased, the needle stick can be seen with a small amount of bleeding, the hair is gradually sparse, and a small amount of secretion is present under the skin flap until the distal color is dark brown. The survival of the skin flaps of group C and D was similar, most of the flaps survived well, the proximal and middle sections were light red, the elasticity was similar to that of the normal tissues, and there was no obvious swelling and swelling. The area of the distal and a few marginal areas, visible part of the brown-black necrotic area, was much smaller than that of group A, group B, acupuncture and hemorrhage, and no apparent accumulation of blood in the skin flap.2. The survival rate of the skin flap: The experimental and statistical analysis showed that the group A (control group) was (57.72-2.03)%. Group B (72.75-0.97)%, group C (81.37-1.49)%, group D (82.31-1.59)%. One-way ANOVA for all data was obtained: P0.05, the total number of data in each group was not completely equal. The data of each group were obtained by means of the LSD method: the experimental group and the control group were compared, and the difference was statistically significant, that is, the survival rate of the skin flap could be increased by subcutaneous injection of rhGH. The results of the comparison between group B and C and D were found in the two groups: PO.05, the difference was statistically significant, and the effect of rhGH on the survival rate of the skin flap was lower than that of the middle and high dose groups. The results showed that there was no significant difference in the survival of the skin flap with high dose of rhGH. No significant tubular vessel, partial fibrous tissue growth. The tissue edema and the infiltration of neutrophils and monocytes in group B were lower than that in group A, and a large number of capillary growth and granulation were clearly visible. The expression of CD34 and VEGF in the tissue was observed by immunohistochemical staining. The results showed that the CD34 in the CD34 stained slide showed brownish yellow particles, which were mainly distributed around the blood capillary of the cytoplasm, and the expression in the group B, C and D was higher than that in group A, the highest in group D and the second in group C. In the expression of VEGF, a large number of brown yellow particles were found in the drug-intervention group, and the control group was in the form of dot-like powder, and the control group was distributed in group B, Group A, Group B, Group B, and the density of microvessel density was detected (MVD): the CD34 immunohistochemical staining slide was placed in the field of 100-fold light microscope. The number of microvessels in 6 fields of view was selected for each slide and the mean value was taken. Group A: (5.00-1.46)/ mm2, group B (10.55-1.78)/ mm2, group C (14.40-1.63)/ mm2, group D (15.19-1.46)/ mm2, and after single-factor analysis of variance, the overall average number of the groups was not exactly equal, that is, the difference between groups was statistically significant. The results showed that there was a significant difference between the drug intervention group and the control group, and the microvessel density of the low-dose group was lower than that of the control group (P0.05). There was no significant difference between the two groups (P0.05). Conclusion:1) The injection of recombinant human growth hormone can promote the expression of CD34 and VEGF in the free flap of the back of the rat, thereby promoting the generation of the new blood vessel in the skin flap;2) the recombinant human growth hormone can improve the survival rate of the free skin flap of the rat; 3) The effect of the recombinant human growth hormone of 0.1-0.2 IU/ kg on the skin flap is the strongest, and can be used as the optimal drug dosage.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622
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相关期刊论文 前10条
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2 林锟;吴涛;罗银燕;;重组人生长激素(安苏萌)对烧伤植皮成活生长的促进作用[J];当代医学;2015年17期
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