外源性VEGF對自體移植脾組織血管再生的影響

1、外源性VEGF對自體移植脾組織血管再生的影響         08-07-09 11:26:00     編輯：studa20            作者：郭光金，蔣登金，姜恒，左艷芳摘 要  目的 探討應(yīng)用外源性VEGF促進(jìn)自體脾組織移植后血管再生與結(jié)構(gòu)重建的影響，為VEGF用于自體移植脾組織的實(shí)驗(yàn)研究和臨床應(yīng)用提供理論參考依據(jù)。方法  

2、;       采用216只昆明種小白鼠隨機(jī)分為脾切除自體脾組織移植組和假手術(shù)組，再將脾移植組分為實(shí)驗(yàn)組和對照組。實(shí)驗(yàn)組小鼠分別于術(shù)后7、14、30、60 d于尾靜脈內(nèi)注射VEGF，注射后7、15、30、60、90 d取脾組織標(biāo)本。進(jìn)行組織學(xué)觀察、血管面密度測定、免疫組化染色方法KDR的檢測、KDR陽性細(xì)胞密度測定、組織原位雜交技術(shù)KDR mRNA檢測。結(jié)果術(shù)后7、15、30 d注射VEGF組  注射VEGF后較對照組的血管數(shù)量明顯增多；術(shù)后60 d注射VEGF組注射VEGF后與對照組各時(shí)相點(diǎn)的組織學(xué)特征沒有明顯差別；術(shù)后7

3、、15、30 d注射VEGF組KDR面積密度值高于對照組，術(shù)后60 d注射VEGF組 KDR面密度值與對照組比較無顯著差別；術(shù)后7、15、30 d注射  KDR mRNA表達(dá)陽性細(xì)胞密度高于對照組，術(shù)后60 d注射VEGF組  KDR mRNA表達(dá)陽性細(xì)胞密度與對照組比較沒有明顯差異。結(jié)論 外源性VEGF能夠促進(jìn)移植脾組織內(nèi)血管生長，自體脾組織移植術(shù)后715 d開始靜脈應(yīng)用VEGF是促進(jìn)血管再生的較理想時(shí)間；外源性VEGF可能是通過與移植脾組織內(nèi)所表達(dá)的KDR結(jié)合發(fā)揮作用。關(guān)鍵詞  自體移植脾組織；血管再生；VEGF；KDR；小鼠   

4、;  Effects of exogenous VEGF on vascular regeneration in autotransplanted splenic tissues of mice Abstract: Objective  To investigate the effects, the feasibility and the mechanism of exogenous VEGF on vascular regeneration and reconstruction of the splenic tissues during the process of sp

5、lenic autotransplantation into the greater omentum. Methods  A total of 216 healthy Kunming mice were randomized into splenic tissue autotransplantation+VEGF group (named as VEGF group), splenic tissue autotransplantation group (control group) and sham operation group. The mice in VEGF group we

6、re injected with exogenous VEGF into the caudal veins on day 7, 15, 30, 60 respectively after operation. Three mice of 3 groups were killed and the splenic tissue was taken as specimen on day 7, 14, 30, 60, 90 after injection of exogenous VEGF to conduct the following investigations. The morphologic

7、 changes in autotransplanted splenic tissue were observed under light microscope and electron microscope. After intubation was conducted through left ventricle and right atria was opened and lavaged with Chinese ink, the splenic tissue pieces were made and the area density of blood vessel was tested

8、 by using computer image system. The density of KDR positive cells was tested by immunohistochemical    methods and computer image analysis. The expression of KDR mRNA were investigated and analyzed by in situ hybridization to study exogenous VEGF on KDR mRNA expression. Results 

9、         On day 7, 15, 30 after injection with exogenous VEGF, the mice in VEGF group were found more renascent blood vessels, splenic corpuscles, central arteries and peripheral lymphatic sheathes in the autotransplanted splenic tissues than the control group

10、, and eventually formed more integral structure of white pulp, marginal zone and red pulp with much less fibrous tissue, while on day 60 there was no significant difference between VEGF group and the control group. On day 7, 15, 30 after injection, the renascent blood vessels were more in number, th

11、e KDR area density and the KDR mRNA expression in splenic tissues were higher in VEGF group than the control group, while on day 60 there were no significant differences in the area density of renascent blood vessel, the KDR area density and the KDR mRNA expression between the two groups. Conclusion

12、  The exogenous VEGF can enhance the vascular regeneration and reconstruction of the autotransplanted splenic tissues into the greater omentum. The day 7 to day 15 after the autotransplantation of splenic tissues was the ideal period for the venous injection of exogenous VEGF that can make its

13、effects through elevating the expression of KDR mRNA.   Keywords: splenic autotransplantation; regeneration; VEGF; KDR; mouse脾臟是人體最大的免疫器官。脾臟損傷或切除后會導(dǎo)致機(jī)體免疫力降低，當(dāng)脾臟嚴(yán)重創(chuàng)傷時(shí)自體脾組織移植手術(shù)已經(jīng)得到廣泛的認(rèn)可，自體移植脾組織通過變性、壞死、再生基本能恢復(fù)原位脾組織的主要結(jié)構(gòu)特征并能夠部分地恢復(fù)其功能。VEGF(血管內(nèi)皮細(xì)胞生長因子) 能與相應(yīng)的受體(KDR) 結(jié)合后有助于新生血管的形成1。因此， 本研究擬靜脈注射外源性

14、VEGF，觀察移植脾組織內(nèi)血管再生與組織結(jié)構(gòu)重建的變化特點(diǎn)，探索應(yīng)用外源性VEGF促進(jìn)移植脾組織內(nèi)血管再生的能力，為臨床提供實(shí)驗(yàn)資料和理論依據(jù)。1  材料和方法1.1  實(shí)驗(yàn)動(dòng)物及分組 健康昆明種小鼠216只(第三軍醫(yī)大學(xué)動(dòng)物所提供)，雌雄不限，體重1525 g，按實(shí)驗(yàn)設(shè)計(jì)分為自體脾組織移植+VEGF組(實(shí)驗(yàn)組) 、自體脾組織移植組(對照組) 和假手術(shù)組。實(shí)驗(yàn)組和對照組均在無菌條件下行脾切除自體脾組織移植術(shù)，取其中間切除脾臟約50%，切成1 mm×1 mm×1mm大小均勻脾組織顆粒，植入大網(wǎng)膜內(nèi)。實(shí)驗(yàn)組于術(shù)后7、15 、30 、60 d從尾靜脈注入VEG

15、F，每組給藥7 d，2次d-1，每次1 ng。注射后7、15、30、60、90 d各處死3只，取移植脾組織標(biāo)本進(jìn)行觀測；假手術(shù)組小鼠僅松動(dòng)脾臟，不行其他手術(shù)處理。1.2 組織形態(tài)學(xué)觀察                                  

16、0;                                               每組按上述時(shí)相點(diǎn)各取3只小鼠，麻醉下放血取

17、脾組織標(biāo)本，常規(guī)福爾馬林固定，石蠟包埋切片，HE染色，光鏡觀察每組動(dòng)物各時(shí)相點(diǎn)的脾組織結(jié)構(gòu)特征。應(yīng)用透射電鏡觀察血管內(nèi)皮細(xì)胞的超微結(jié)構(gòu)特征。1.3 血管密度測定                                    

18、0;                                             每組各時(shí)相點(diǎn)分別取小鼠3只，經(jīng)左心室行主動(dòng)脈插管，灌注墨汁福爾馬林

19、混懸液，直至小鼠唇、肢端變黑后，切取移植脾組織標(biāo)本，石蠟包埋切片，進(jìn)行計(jì)算機(jī)圖象分析測定血管面密度。1.4  KDR的檢測                                       

20、                                           每組各時(shí)相點(diǎn)分別取脾切除自體脾組織移植組小鼠3只及對照組小鼠3只，腹腔注射麻醉，取脾組織福爾馬

21、林固定，常規(guī)石蠟包埋、切片，免疫組化ABC法抗KDR抗體染色，光鏡觀察，采用醫(yī)學(xué)圖象分析系統(tǒng)，對不同時(shí)相點(diǎn)免疫組織化學(xué)KDR面密度。1.5 KDR mRNA的原位雜交檢測