不同劑量重組人生長(zhǎng)激素皮下注射對(duì)大鼠背部隨意皮瓣存活影響的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2019-06-17 09:13
【摘要】：研究背景:隨著對(duì)創(chuàng)面修復(fù)的不斷深入研究,越來(lái)越多的目光轉(zhuǎn)向皮瓣修復(fù)。這種修復(fù)方法不僅能完成對(duì)創(chuàng)面的完全覆蓋,還能解決創(chuàng)基薄弱、骨及肌腱外露等傳統(tǒng)手術(shù)修復(fù)困難的難治性創(chuàng)面,因此,各大外科對(duì)皮瓣修復(fù)的應(yīng)用也日益頻繁。而隨意皮瓣作為其中最常用方法之一,其成活率的提高一直廣受醫(yī)生、學(xué)者關(guān)注和研究。隨意皮瓣內(nèi)缺少知名血管及缺血-再灌注損傷(I-R損傷)仍極大的限制著隨意皮瓣的成活。近年來(lái),隨著皮瓣設(shè)計(jì)的不斷完善,比如:最佳長(zhǎng)寬比例、皮瓣形狀、術(shù)中不同血管吻合方式、延遲行皮瓣移植、加強(qiáng)創(chuàng)面護(hù)理、術(shù)后定期高壓氧治療以及更換敷料時(shí)各種生長(zhǎng)因子的應(yīng)用等方法的大量推行,皮瓣成活率顯著上升,但臨床應(yīng)用中皮瓣壞死的情況仍屢見(jiàn)不鮮。如何能最大程度避免皮瓣中I-R損傷的發(fā)生,提高其皮瓣生存率成為近年來(lái)的熱門(mén)研究方向。重組人生長(zhǎng)激素(rhGH)在以往研究中已被證實(shí)了有增強(qiáng)血漿蛋白mRNA表達(dá)和促進(jìn)血清白蛋白(ALB)合成、提高血糖、分解脂質(zhì)、免疫調(diào)控、增生血管、減少蛋白分解,減少氮丟失,促進(jìn)傷口愈合的作用。在此基礎(chǔ)之上,rhGH的臨床應(yīng)用不斷增加,并擴(kuò)展到燒傷創(chuàng)面及皮瓣存活方面。皮瓣移植后補(bǔ)充rhGH可以明顯降低炎癥因子白細(xì)胞介素-1(IL-1)、白細(xì)胞介素-6(IL-6)、腫瘤壞死因子-a(TNF-a)的表達(dá),減少中性粒細(xì)胞的聚集。并刺激單核細(xì)胞的遷移、促進(jìn)超氧陰離子生成,使機(jī)體的抵抗力得以提高,從而減輕炎癥反應(yīng)。彭麗霞等研究表明,大鼠皮下注射rhGH后可促進(jìn)皮瓣中血管內(nèi)皮生長(zhǎng)因子(VEGF)的表達(dá),從而起到促進(jìn)血管新生的作用,使皮瓣壞死率降低。但其中劑量的選擇尚未進(jìn)行明確實(shí)驗(yàn)證明�；谏鲜鲈�,本次實(shí)驗(yàn)以大鼠為模型,驗(yàn)證不同劑量rhGH對(duì)大鼠背部隨意皮瓣的影響,以了解其皮下注射的有效劑量。目的:通過(guò)皮下注射rhGH影響大鼠背部隨意皮瓣成活,并采用不同注射劑量,觀察不同劑量的rhGH對(duì)大鼠背部隨意皮瓣影響程度,找出最佳注射劑量,為其臨床應(yīng)用前景做出初步評(píng)估。方法:健康成年SD大鼠64只,均在其背部正中設(shè)計(jì)蒂在尾端的大小為2cm*4cm的隨意皮瓣,并采用隨機(jī)分配原則,將大鼠分為A(生理鹽水組)、B(0.05IU/Kg rhGH組)、C(0.1 IU/Kg rhGH組)、D(0.2 IU/Kg rhGH組)四組。術(shù)后即刻開(kāi)始皮下注射藥物,連續(xù)7天,一日一次。四組均于術(shù)后第7天拍照計(jì)算皮瓣成活率,并取所有皮瓣遠(yuǎn)端進(jìn)行實(shí)驗(yàn)結(jié)果觀察。將標(biāo)本進(jìn)行HE染色、免疫組化染色觀察VEGF及CD34在組織中的表達(dá)情況,計(jì)算皮瓣微血管密度(MVD),數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析,明確實(shí)驗(yàn)?zāi)康摹＝Y(jié)果:1、皮瓣大體觀察:A組皮瓣近端皮瓣成活區(qū)呈粉紅色,毛發(fā)稀疏。中段及遠(yuǎn)端部分壞死,呈黑色,質(zhì)硬,彈性明顯降低,無(wú)毛發(fā)生長(zhǎng),針刺不出血,皮瓣下淡紅色分泌物較多。B組皮瓣近端色澤紅潤(rùn),成活尚可,中段開(kāi)始逐漸色澤晦暗,呈棕色,少量黑色痂殼附著,彈性降低,針刺可見(jiàn)少量出血,毛發(fā)逐漸稀疏,皮瓣下存在少量分泌物,直至遠(yuǎn)端色棕黑。C組及D組皮瓣成活情況相似,大部分皮瓣存活良好,近端及中段呈淡紅色,彈性與正常組織相似,無(wú)明顯紅腫等情況。遠(yuǎn)端及少許邊緣部位,可見(jiàn)部分棕黑色壞死區(qū),面積遠(yuǎn)小于A、B組,針刺出血,皮瓣下無(wú)明顯積血積液。2、皮瓣成活率:經(jīng)實(shí)驗(yàn)及統(tǒng)計(jì)學(xué)分析可以得出:A組(對(duì)照組)為(57.72±2.03)%,B組為(72.75±0.97)%,C組為(81.37±1.49)%,D組為(82.31±1.59)%。將全部數(shù)據(jù)進(jìn)行單因素方差分析(one-way ANOVA)得出:P0.05,各組數(shù)據(jù)總體均數(shù)不全相等。再運(yùn)用LSD法將各組數(shù)據(jù)進(jìn)行兩兩比較可得:各實(shí)驗(yàn)組與對(duì)照組進(jìn)行比較,PO.05,差異有統(tǒng)計(jì)學(xué)意義,即rhGH皮下注射可提高皮瓣成活率。B組與C、D兩組比較結(jié)果可見(jiàn):PO.05,差異有統(tǒng)計(jì)學(xué)意義,可得出低劑量組rhGH對(duì)皮瓣成活率的促進(jìn)作用低于中、高劑量組;C、D組兩組統(tǒng)計(jì)學(xué)比較得出PO.05,差異無(wú)統(tǒng)計(jì)學(xué)意義,故中、高劑量rhGH促進(jìn)皮瓣成活無(wú)顯著性差異。3、HE染色可見(jiàn):A組鏡下見(jiàn)組織結(jié)構(gòu)混亂不清,各層結(jié)構(gòu)顯示不明,組織明顯水腫,大量中性粒細(xì)胞及單核細(xì)胞浸潤(rùn),呈急性炎癥反應(yīng)改變,少量薄壁血管生長(zhǎng),無(wú)明顯管狀血管,部分纖維組織生長(zhǎng)。B組真皮下組織較混亂,組織水腫及中性粒細(xì)胞及單核細(xì)胞浸潤(rùn)較A組減輕,大量毛細(xì)血管生長(zhǎng),肉芽清晰可見(jiàn)。C組及D組鏡下少許炎癥細(xì)胞浸潤(rùn),組織結(jié)構(gòu)顯示清晰,新生管狀血管生成,內(nèi)可見(jiàn)紅細(xì)胞充盈。4、CD34及VEGF在組織中的表達(dá):運(yùn)用免疫組化染色觀察組織內(nèi)CD34及VEGF的表達(dá)強(qiáng)弱,結(jié)果明確可見(jiàn)CD34染色玻片中CD34呈棕黃色顆粒,主要分布于細(xì)胞漿毛細(xì)血管周?chē)?在B、C、D組中的表達(dá)明顯高于A組,D組最高,C組其次。而VEGF免疫組化檢測(cè)中呈藥物干預(yù)組可見(jiàn)大量棕黃色顆粒分布成團(tuán)狀,對(duì)照組則呈點(diǎn)狀散在分布,分布密度D組≥C組B組A組。5、微血管密度檢測(cè)(MVD):將CD34免疫組化染色玻片置于100倍光鏡視野下,每張玻片選取6個(gè)視野計(jì)數(shù)微血管數(shù)目并取其均值。A組:(5.00±1.46)條/mm2,B組(10.55±1.78)條/mm2,C組(14.40±1.63)條/mm2,D組(15.19±1.46)條/mm2,進(jìn)行單因素方差分析后得出,P0.05,各組總體均數(shù)不完全相等,即表示各組間差異有統(tǒng)計(jì)學(xué)意義。再LSD法兩兩比較得出,藥物干預(yù)組與對(duì)照組P0.05,有顯著差異,C、D組與B組比較得出P0.05,低劑量組微血管密度偏低。C、D兩組比較P0.05,差異無(wú)統(tǒng)計(jì)學(xué)意義。結(jié)論:1)重組人生長(zhǎng)激素皮下注射可促進(jìn)大鼠背部隨意皮瓣內(nèi)CD34及VEGF的表達(dá)增加,從而促進(jìn)皮瓣內(nèi)新生血管生成;2)重組人生長(zhǎng)激素能提高大鼠隨意皮瓣的成活率;3)0.1-0.2IU/kg劑量的重組人生長(zhǎng)激素對(duì)皮瓣的作用最強(qiáng),可作為最佳藥物劑量使用。
[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.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：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.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R622
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