【摘要】：目的:1、探討PA的T3SS結(jié)構(gòu)對(duì)糖尿病潰瘍創(chuàng)面組織中自噬相關(guān)蛋白表達(dá)的影響,并進(jìn)一步探討其對(duì)糖尿病潰瘍創(chuàng)面愈合的影響。2、探討PA的T3SS分泌的毒力蛋白ExoU、ExoS分別對(duì)糖尿病潰瘍創(chuàng)面組織中自噬相關(guān)蛋白表達(dá)的影響,并進(jìn)一步探討其對(duì)糖尿病潰瘍創(chuàng)面愈合的影響。3、探討雷帕霉素及慶大霉素對(duì)PA感染的糖尿病潰瘍創(chuàng)面組織中自噬相關(guān)蛋白表達(dá)的影響及對(duì)糖尿病潰瘍創(chuàng)面愈合的影響。方法:一、第一部分:36只SD大鼠隨機(jī)分為六組:糖尿病非感染組(DM+未感染),糖尿病感染PAK組(DM+PAK),糖尿病感染PAK△pcrV-(DM+PAK△pcrV-),非糖尿病非感染組(N+未感染),非糖尿病感染PAK組(N+PAK),非糖尿病感染PAK△pcrV-(N+PAK△pcrV-),每組各6只。分別在造模第0天、3天、7天、14天、21天觀察創(chuàng)面愈合情況并進(jìn)行創(chuàng)面內(nèi)菌落計(jì)數(shù);分別在造模第0、7、14天取創(chuàng)面組織,HE染色觀察各組大鼠創(chuàng)面組織內(nèi)細(xì)胞形態(tài)、結(jié)構(gòu)變化;免疫組化及Western blotting檢測(cè)各組大鼠創(chuàng)面組織內(nèi)自噬相關(guān)蛋白LC3、Beclin-1、p62的表達(dá);免疫熒光雙染色檢測(cè)各組大鼠創(chuàng)面組織內(nèi)巨噬細(xì)胞中LC3的表達(dá);二、第二部分:24只SD大鼠隨機(jī)分為四組:糖尿病感染PA103組(DM+PA103),糖尿病感染PA01(DM+PA01),非糖尿病感染PA103組(N+PA103),非糖尿病感染PA01(N+PA01),每組各6只。方法同第一部分;三、第三部分:36只SD大鼠隨機(jī)分為六組:糖尿病感染PA103組(DM+PA103),糖尿病感染PA103且雷帕霉素干預(yù)組(DM+PA103+雷帕霉素),非糖尿病感染PA103組(N+PA103),非糖尿病感染PA103且雷帕霉素干預(yù)組(N+PA103+雷帕霉素),糖尿病感染PA103且慶大霉素干預(yù)組(DM+PA103+慶大霉素),非糖尿病感染PA103且慶大霉素干預(yù)組(N+PA103+慶大霉素),每組各6只。方法同第一部分。結(jié)果:一、第一部分:1、創(chuàng)面愈合情況:與非糖尿病組大鼠相比,糖尿病組大鼠第3天有黑痂形成,第7天有較多膿性分泌物,創(chuàng)面未見縮小,第21天創(chuàng)面仍未愈合,傷愈合時(shí)間明顯延遲。在糖尿病組中,與DM+未感染及DM+PAK△pcrV-相比,第3天DM+PAK組黑痂形成、壞死較嚴(yán)重、周圍較紅腫,第7天有膿性分泌物滲出且最多,第21天仍有較多分泌物,第21傷口直徑較大。2、創(chuàng)面內(nèi)菌落計(jì)數(shù):與非糖尿病感染組相比,糖尿病感染組菌落增長(zhǎng)速度較快且減少速度較慢,其中與DM+PAK△pcrV-相比,DM+PAK組第7天以后菌落數(shù)開始減少,但減少不明顯。3、HE結(jié)果:糖尿病感染組在第7、14天炎性粒細(xì)胞浸潤(rùn)逐漸增加,與DM+PAK△pcrV-相比,DM+PAK分別在第7、14天有更多炎性細(xì)胞浸潤(rùn)。非糖尿病組感染組在第7天炎性細(xì)胞浸潤(rùn)達(dá)到高峰,第14天炎性細(xì)胞下降,與N+PAK△pcrV-組相比,N+PAK分別在第7、14天有更多炎性細(xì)胞浸潤(rùn)。4、免疫組化結(jié)果:各組LC3、Beclin-1在第0、7、14天表達(dá)遞增,p62蛋白第7、14天表達(dá)均逐漸下降(P均0.05)。N+非感染組LC3、Beclin-1表達(dá)在第7、14天表達(dá)明顯高于DM+非感染組,p62表達(dá)在第7、14天低于DM+非感染組(P均0.05)。DM+PAK△pcrV-組LC3、Beclin-1表達(dá)在第14天高于DM+PAK,p62表達(dá)在第14天低于DM+PAK(P均0.05);N+PAK△pcrV-組中LC3、Beclin-1表達(dá)在第7、14天高于N+PAK,p62表達(dá)在第7、14天低于N+PAK(P均0.05);與N+PAK組相比,DM+PAK組在第14天LC3、Beclin-1表達(dá)較低,p62表達(dá)較高(P均0.05);與N+PAK△pcrV-組相比,DM+PAK組LC3、Beclin-1表達(dá)在第7、14天較低,p62表達(dá)在第7、14天較高(P均0.05)。5、免疫印跡結(jié)果:同免疫組化結(jié)果相一致。6、免疫熒光結(jié)果:DM+PAK△pcrV-組巨噬細(xì)胞中LC3表達(dá)在第14天高于DM+PAK組;N+PAK△pcrV-組中巨噬細(xì)胞中LC3表達(dá)在第7、14天高于N+PAK。二、第二部分:1、創(chuàng)面愈合情況:與DM+PA01相比,DM+103組第3天有黑痂形成、壞死較嚴(yán)重、周圍較紅腫,傷口直徑擴(kuò)大,第14天傷口直徑仍未縮小,壞死仍嚴(yán)重,第21天仍有較多分泌物,創(chuàng)面仍未愈合。2、創(chuàng)面內(nèi)菌落計(jì)數(shù):DM+PA103和DM+PA01在第0-7天持續(xù)增長(zhǎng),與DM+PA01相比,DM+PA103增加較快,第7天以后菌落數(shù)開始減少,減少較緩慢。3、HE結(jié)果:與DM+PA01組相比,DM+PA103分別在第7、14天有較多中性粒細(xì)胞、巨噬細(xì)胞浸潤(rùn)。4、免疫組化結(jié)果:各組LC3、Beclin-1在第0、7、14天表達(dá)遞增,p62蛋白第7、14天表達(dá)均逐漸下降(P均0.05)。DM+PA103組LC3、Beclin-1表達(dá)在第14天低于DM+PA01,p62表達(dá)在第14天高于DM+PA01(P均0.05);N+PA103組LC3、Beclin-1表達(dá)在第7天低于N+PA01,p62表達(dá)在第7天高于N+PA01(P均0.05);與N+103組相比,DM+103組LC3、Beclin-1表達(dá)在第7、14天較低,p62表達(dá)在第7、14天較高(P均0.05)。5、免疫印跡結(jié)果:同免疫組化結(jié)果。6、免疫熒光結(jié)果:DM+103組巨噬細(xì)胞中LC3表達(dá)在第14天低于DM+PA01;N+PA01組巨噬細(xì)胞中LC3表達(dá)在第7天高于N+PA103。三、第三部分:1、創(chuàng)面愈合情況:與DM+PA103相比,DM+PA103+雷帕霉素在第7天膿性分泌物較前減少,第14天創(chuàng)面直徑明顯縮小。DM+PA103+慶大霉素組在第7天創(chuàng)面直徑明顯縮小,第21天傷口趨于愈合。與N+PA103相比,N+PA103+雷帕霉素在第3天組織破潰較輕,膿性分泌物滲出較少,第7天創(chuàng)面直徑明顯縮小,第21天創(chuàng)面基本愈合。N+PA103+慶大霉素組第3天結(jié)痂較輕,第21天創(chuàng)面基本愈合。2、創(chuàng)面內(nèi)菌落計(jì)數(shù):DM+PA103和DM+PA103+雷帕霉素在第0-7天持續(xù)增長(zhǎng),與DM+PA103組相比,DM+PA103+雷帕霉素增加較慢,第7天以后菌落數(shù)減少較快,DM+PA103+慶大霉素組第7天菌落數(shù)減少較快;與N+PA103組相比,N+PA103+雷帕霉素在第3天后菌落明顯減少,N+PA103+慶大霉素第3天后菌落明顯減少(P0.05)。3、HE結(jié)果:與DM+PA103組相比,DM+PA103+雷帕霉素與DM+PA103+慶大霉素組分別在第7、14天中性粒細(xì)胞、巨噬細(xì)胞浸潤(rùn)等炎癥細(xì)胞數(shù)目較少。與N+PA103組相比,N+PA103+雷帕霉素與N+PA103+慶大霉素分別在第7、14天炎癥細(xì)胞浸潤(rùn)減少。4、免疫組化結(jié)果:各組LC3、Beclin-1在第0、7、14天表達(dá)遞增,p62蛋白第7、14天表達(dá)均逐漸下降(P均0.05)。DM+PA103+雷帕霉素組LC3表達(dá)在第7、14天高于DM+PA103,Beclin-1表達(dá)在第14天高于DM+PA103,p62表達(dá)在7、14低于N+PA103(P均0.05);N+PA103+雷帕霉素組LC3在第7、14天高于DM+PA103,Beclin-1表達(dá)在第7天高于N+PA103,p62表達(dá)在第7天低于N+PA103(P均0.05);與DM+PA103+雷帕霉素組相比,N+PA103+雷帕霉素組LC3、Beclin-1表達(dá)在第7、14天較高,p62表達(dá)在第7、14天較高(P均0.05)。DM+PA103+慶大霉素組和DM+PA103組在各個(gè)時(shí)間點(diǎn)Beclin-1、LC3、p62的表達(dá)量均無(wú)顯著差異,N+PA103+慶大霉素組與N+PA103組在各個(gè)時(shí)間點(diǎn)Beclin-1、LC3、p62的表達(dá)量均無(wú)顯著差異(P均0.05)。5、免疫印跡結(jié)果:同免疫組化。6、免疫熒光結(jié)果:DM+PA103+雷帕霉素組巨噬細(xì)胞中LC3表達(dá)在第7、14天高于DM+PA103;N+PA103+雷帕霉素組巨噬細(xì)胞中LC3在第7、14天高于N+PA103。結(jié)論:1、在創(chuàng)面愈合過(guò)程中,非糖尿病及糖尿病大鼠創(chuàng)面組織中細(xì)胞自噬蛋白水平均增強(qiáng),但是糖尿病組的自噬水平明顯低于非糖尿病組,且糖尿病創(chuàng)面炎癥持續(xù),創(chuàng)面愈合延遲。2、在糖尿病感染創(chuàng)面中,PA的T3SS均可抑制自噬蛋白水平的表達(dá),自噬對(duì)細(xì)菌的清除能力減弱,PA的T3SS分泌的毒力蛋白ExoU抑制自噬蛋白水平的表達(dá)更明顯,自噬對(duì)細(xì)菌的清除能力明顯減弱,ExoU可導(dǎo)致糖尿病創(chuàng)面壞死更嚴(yán)重,感染持續(xù)且糖尿病創(chuàng)面愈合受損。3、在非糖尿病感染創(chuàng)面和糖尿病感染創(chuàng)面中,雷帕霉素可通過(guò)增強(qiáng)自噬促進(jìn)對(duì)PA的清除可加速創(chuàng)面愈合,但其在糖尿病狀態(tài)的效果弱于非糖尿病狀態(tài)。4、慶大霉素在感染病創(chuàng)面可有效地清除PA以減少感染,進(jìn)而促進(jìn)創(chuàng)愈合,無(wú)論在糖尿病創(chuàng)面還是非糖尿創(chuàng)面,慶大霉素對(duì)創(chuàng)面內(nèi)自噬蛋白水平無(wú)明顯影響。
[Abstract]:Objective: 1, to investigate the effect of T3SS structure of PA on the expression of autophagy related protein in the tissue of diabetic ulcer wound, and further explore the effect of.2 on the healing of diabetic ulcer wound, and explore the effect of ExoS on the expression of autophagy related protein in the tissue of diabetic ulcer wound, and further explore the effect of ExoS on the expression of autophagic protein in the tissue of diabetic ulcer wound. Effect of.3, the effect of rapamycin and gentamicin on the expression of autophagy related protein in the tissue of diabetic ulcer wound with PA infection and the effect on the healing of diabetic ulcer wound. Methods: 1. Part one: 36 SD rats were randomly divided into six groups: diabetes non infected group (DM+ uninfected), diabetes sense PAK group (DM+PAK), diabetes infection PAK Delta pcrV- (DM+PAK Delta pcrV-), non diabetic non infection group (N+ uninfected), non diabetic PAK group (N+PAK), non diabetic PAK Delta pcrV- (N+PAK delta), each group of 6 rats, respectively in the model zeroth days, 3 days, 7 days, 14 days, 21 days to observe the wound healing and to carry on the count of bacterial colonies in the wound; respectively The wound tissue was taken on day 0,7,14, and the morphological and structural changes in the wound tissue were observed by HE staining. Immunohistochemistry and Western blotting were used to detect the expression of autophagy related protein LC3, Beclin-1, p62 in the wound tissue of each group, and the expression of LC3 in the macrophages in the wound tissue of the rats was detected by immunofluorescence; two The two part: 24 SD rats were randomly divided into four groups: diabetic PA103 group (DM+PA103), diabetes infected with PA01 (DM+PA01), non diabetic infection PA103 group (N+PA103), non diabetic infection PA01 (N+PA01), each group with the first part; three, third parts: 36 SD rats were randomly divided into six groups: diabetes PA103 group (DM+PA103), diabetes mellitus Disease infection PA103, rapamycin intervention group (DM+PA103+ rapamycin), non diabetic PA103 group (N+PA103), non diabetic PA103 and rapamycin intervention group (N+PA103+ rapamycin), diabetes infection PA103 and gentamicin intervention group (gentamicin), non diabetic infection PA103 and gentamicin intervention group (N+PA103+ celebration group) 6 in each group. Methods and the first part. Results: first, part one: 1, wound healing: compared with the non diabetic rats, the rats in the diabetic group had black eschar formation on third days, there were more purulent secretions on seventh days, the wound was not reduced, the wounds were still not healed on the twenty-first day, and the healing time was delayed. In the diabetic group, the DM+ was not felt with the diabetic group. Compared with DM+PAK Delta pcrV-, the third day group of DM+PAK group black scab formed, the necrosis was more serious, the surrounding was more red and swelling, the purulent secretions were exudative and most in the seventh day, there were more secretions in twenty-first days, the twenty-first wound diameter was larger than that of the non diabetic infection group, and the growth rate of the diabetic infection group was faster and less speed than that of the non diabetic infection group. As compared with DM+PAK Delta pcrV-, the colony number of the DM+PAK group began to decrease after seventh days, but the decrease was not obvious.3. The HE result: the inflammatory granulocyte infiltration in the diabetic infection group gradually increased on the 7,14 day, and the DM+PAK was more inflammatory cell infiltration in the 7,14 day, compared with DM+PAK Delta pcrV-. Up to the peak, the fourteenth days of inflammatory cells decreased. Compared with the N+PAK Delta pcrV- group, N+PAK had more inflammatory cells infiltrating.4 on day 7,14. The results of immunohistochemistry: LC3, Beclin-1 in 0,7,14 days were increased, and the expression of p62 protein in 7,14 day decreased gradually (P was 0.05). In the non infected DM+ group, the expression of p62 was lower than the DM+ non infection group (P 0.05).DM+PAK Delta pcrV- group LC3, the Beclin-1 expression was higher than DM+PAK in the fourteenth day, and the p62 expression was lower than DM+PAK (all 0.05) at fourteenth days. In group DM+PAK, the expression of Beclin-1 was lower at fourteenth days and the expression of p62 was higher (P 0.05). Compared with N+PAK Delta pcrV- group, DM+PAK group LC3, Beclin-1 expression was lower in 7,14 day, p62 expression was higher (0.05). Immunoblotting results: immunoblotting results: immunofluorescence results: expression of immunofluorescence On the fourteenth day higher than the DM+PAK group, the expression of LC3 in the macrophages in the N+PAK Delta pcrV- group was higher than N+PAK. two, the second part: 1, the wound healing. Compared with DM+PA01, the DM+103 group had black eschar formation on third days, the necrosis was more severe, the diameter of the wound was more red, the diameter of the wound was enlarged, the diameter of the wound was still not narrowed, the necrosis was still serious and twenty-first days was still more serious. .2 was still not healed in the wound, and the colony count in the wound was not healed. DM+PA103 and DM+PA01 continued to increase on day 0-7. Compared with DM+PA01, DM+PA103 increased rapidly. After seventh days the colony number began to decrease, and the slow.3, HE result: DM+PA103 was more neutrophils in 7,14 day than DM+PA01 group, and macrophage infiltration was.4, immunization. The expression of LC3 and Beclin-1 increased gradually on day 0,7,14. The expression of p62 protein on day 7,14 decreased gradually (P 0.05).DM+PA103 group LC3, Beclin-1 expression was lower than DM+PA01 in the fourteenth day, p62 expression was higher in Fourteenth days than DM+PA01 (0.05). Compared with the N+103 group, the expression of LC3 and Beclin-1 in group DM+103 was lower on day 7,14, p62 expression was higher on day 7,14 (P 0.05).5, and immunoblotting results: the result of immunoblotting.6, immunofluorescence results: LC3 expression in the DM+103 group macrophages was lower than three, third part of macrophage in seventh days. 1, wound healing: compared with DM+PA103, the seventh days of rapamycin was reduced in seventh days, the diameter of the wound was significantly reduced in Fourteenth days and the diameter of.DM+PA103+ gentamicin group narrowed at seventh days, and the wounds tended to heal in twenty-first days. Compared with N+PA103, N+ PA103+ rapamycin had a lighter tissue break and a purulent secretion in the third day. The exudation was less, the diameter of the wound was obviously reduced in seventh days, the basic healing of the wound was twenty-first days in the group of.N+PA103+ gentamicin group, and the wound healing was lighter in third days. The wound healing was basically.2 in the twenty-first day. The number of DM+PA103 and DM+PA103+ rapamycin continued to increase on the 0-7 day, compared with the DM+PA103 group, the increase of DM+PA103+ rapamycin was slower, and seventh days later, the bacteria were increased. The colony number of DM+PA103+ gentamicin group decreased faster in the seventh day of gentamicin group. Compared with group N+PA103, the colony of N+PA103+ rapamycin decreased obviously after third days, and the colony of N+PA103+ gentamicin decreased significantly (P0.05).3, HE result: DM+ PA103+ rapamycin and DM+PA103+ gentamicin group were in the first 7,14 days, respectively. The number of inflammatory cells in neutrophils and macrophage infiltration was less. Compared with the N+PA103 group, the infiltration of N+PA103+ rapamycin and N+PA103+ gentamicin decreased.4 respectively on day 7,14, and the immunohistochemical results showed that the expression of LC3, Beclin-1 in 0,7,14 days increased, and the expression of p62 egg white in 7,14 day decreased gradually (P all 0.05).DM+PA103+ Lei The expression of LC3 in the paramiomycin group was higher in the day 7,14 than in DM+PA103, and the expression of Beclin-1 was higher in Fourteenth days than in DM+PA103, and the expression of p62 in 7,14 was lower than N+PA103 (P 0.05); N+PA103+ rapamycin group was higher in 7,14 day than in seventh days. The expression of LC3 and Beclin-1 in +PA103+ rapamycin group was higher on 7,14 day, p62 expression was higher on 7,14 day (P all 0.05).DM+PA103+ gentamicin group and DM+PA103 group at all time points Beclin-1, LC3, p62 expression was not significant difference. Difference (P 0.05).5, immunoblotting results: immunofluorescence.6, immunofluorescence results: the expression of LC3 in macrophages in DM+PA103+ rapamycin group was higher in 7,14 day than DM+PA103; LC3 in N+PA103+ of rapamycin group was higher in N+PA103. conclusion on 7,14 day: 1, in wound healing process, the wound tissue of non diabetic and diabetic rats was fine. The level of autophagic protein was enhanced, but the level of autophagy in the diabetic group was significantly lower than that in the non diabetic group, and the inflammation of the diabetic wound continued and the wound healing delayed.2. In the diabetic infection wound, PA T3SS could inhibit the expression of autophagic protein, the ability to remove autophagy to bacteria and the inhibitory protein ExoU secreted by PA T3SS. The expression of autophagic protein is more obvious, the ability of autophagy to scavenge bacteria is significantly weakened, ExoU can lead to more severe diabetic wound necrosis, continuous infection and impaired healing of diabetic wound.3. In non diabetic wound and diabetic infection wounds, rapamycin can accelerate wound healing by enhancing autophagy to promote the clearance of PA. However, its effect on diabetes is weaker than non diabetic.4. Gentamicin can effectively remove PA to reduce infection and thus promote healing. Gentamicin has no significant effect on the level of autophagic protein in wound and non diabetic wound.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2

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