本文選題：大鼠 + 皮瓣��； 參考：《浙江大學(xué)》2014年博士論文

【摘要】：背景 皮瓣手術(shù)是整形重建和口腔頜面部手術(shù)中最重要的技術(shù)之一。然而,無論是在實(shí)驗(yàn)還是臨床中,外科皮瓣壞死都是很難完全預(yù)防和避免的。皮瓣壞死往往導(dǎo)致皮瓣摘除。很多的研究都嘗試通過藥物,提高皮瓣成活率。然而,在這些研究中也有明顯的一些不足。許多研究中沒有動物模型解剖學(xué)證據(jù)；當(dāng)壞死即將發(fā)生時,沒有很好的解決方法；雖然一直對壞死的預(yù)防性研究很多,但關(guān)于促進(jìn)壞死修復(fù)的研究很少,對皮瓣壞死創(chuàng)口治療的研究更為鮮見。 KGF、EGF和VEGF是生長因子領(lǐng)域研究最多的幾個生長因子,大量的文獻(xiàn)都確定其分別特異性對上皮細(xì)胞、主要對上皮細(xì)胞、特異性對血管內(nèi)皮細(xì)胞有很強(qiáng)的生長促進(jìn)作用。KGF對上皮細(xì)胞的特異性作用,使其能促進(jìn)上皮細(xì)胞增殖的同時,不促進(jìn)纖維生長,不導(dǎo)致疤痕形成,成為促進(jìn)上皮創(chuàng)口愈合最好的候選。EGF是臨床上應(yīng)用最多的生長因子之一,其對上皮的促進(jìn)作用得到很多患者及醫(yī)生的認(rèn)同。VEGF是迄今為止發(fā)現(xiàn)的促進(jìn)血管新生最有效的生長因子,能夠極大地提高外科皮瓣的成活,預(yù)防皮瓣壞死。 皮瓣壞死的基因預(yù)防及治療是一種極有潛力的方法,是未來研究的發(fā)展方向。當(dāng)今皮瓣的基因研究主要是以生長因子作為治療性基因完成的。如何以最佳的方法將外源性DNA導(dǎo)入皮瓣,使細(xì)胞接收并表達(dá)外源DNA,一直是值得大家研究和探討的問題。 我們以腺病毒為載體(Ad),攜帶角化細(xì)胞生長因子(KGF)基因的方法,利用復(fù)制缺陷型腺病毒載體重組KGF基因,通過其強(qiáng)感染能力與轉(zhuǎn)基因能力,在體外體內(nèi)特異性表達(dá)有活性的KGF蛋白,發(fā)揮定向修復(fù)組織損傷的作用。另外我們直接在大鼠背部的壞死皮瓣區(qū)域中和皮膚中注入EGF和VEGF,觀察聯(lián)合應(yīng)用生長因子是否會對皮瓣區(qū)壞死愈合起作用。 方法 1.Pac Ⅰ核酸內(nèi)切酶酶切鑒定腺病毒質(zhì)粒,測序檢測是否質(zhì)粒存在變異。Pac Ⅰ核酸內(nèi)切酶酶切pAd-KGF,線性化后的pAd-KGF轉(zhuǎn)染進(jìn)HEK293(293)細(xì)胞后,制備Ad-KGF。應(yīng)用293細(xì)胞擴(kuò)增、純化得到Ad-KGF病毒。應(yīng)用于后續(xù)細(xì)胞及動物實(shí)驗(yàn),并觀察病毒轉(zhuǎn)染293細(xì)胞后,細(xì)胞形態(tài)及病毒的感染能力。 2.Ad-KGF感染正常細(xì)胞系PA317,HUVEC,HaCaT,和293細(xì)胞后,觀察病毒的感染的能力；提取受感染細(xì)胞的RNA,逆轉(zhuǎn)錄得到cDNA后,進(jìn)行半定量PCR和熒光定量PCR,檢測細(xì)胞內(nèi)KGF的表達(dá)水平；通過ELISA,檢測受感染細(xì)胞上清中KGF的表達(dá)水平；通過劃痕實(shí)驗(yàn)?zāi)７聜?培養(yǎng)HaCaT細(xì)胞48小時,確定KGF對上皮細(xì)胞生長遷移的作用；將PA317細(xì)胞,HUVEC細(xì)胞和HaCaT細(xì)胞接種于Transwell小室,293細(xì)胞+Ad-KGF轉(zhuǎn)染24小時(293+Ad-KGF),293細(xì)胞+Ad轉(zhuǎn)染24小時(293+Ad),HaCaT,293細(xì)胞和293細(xì)胞+KGF(293+KGF)接種于下室內(nèi),觀察KGF和Ad-KGF對成纖維細(xì)胞系,血管內(nèi)皮細(xì)胞系和上皮細(xì)胞系的生長遷移作用。 3.我們首先解剖大鼠背部,研究確定大鼠背部血管走行。在傳統(tǒng)大鼠背部皮瓣模型上,改良手術(shù)及實(shí)驗(yàn)過程,使皮瓣發(fā)生壞死。記錄大鼠的體重,皮瓣壞死面積和愈合情況。在術(shù)后第15天,25天,和45天過量水合氯醛處死大鼠,取背部壞死皮瓣標(biāo)本,進(jìn)行組織化學(xué)染色分析。 4.在建立大鼠背部皮瓣模型之后,所有動物分為四組。將1ml PBS+dexamethasone(DXM),1ml PBS+Ad-KGF+DXM,or1ml PBS+Ad+DXM分別注入大鼠背部皮瓣壞死區(qū)皮下和創(chuàng)口邊緣,分別于術(shù)后5天,10天,20天,和30天注射相同的藥物。隔日記錄體重,直至創(chuàng)面壞死愈合。術(shù)后第15天,25天,和35天過量水合氯醛處死大鼠,取背部壞死皮瓣標(biāo)本,進(jìn)行組織化學(xué)Masson染色、免疫組化染色分析和熒光定量PCR分析,上皮及上皮下組織的生長情況,上皮的生長厚度,取血清進(jìn)行ELISA檢測KGF表達(dá)水平。 5.首先通過細(xì)胞遷移實(shí)驗(yàn)確定EGF和VEGF對PA317,HUVEC,和HaCaT細(xì)胞遷移的活性。建立大鼠背部皮瓣模型,壞死發(fā)生后,沿創(chuàng)口邊緣及壞死皮下注入1ml PBS+EGF (150mg/kg),1ml PBS+VEGF (10μg), or1ml PBS+EGF (150μg/kg)+VEGF(10μg)。隔日記錄體重并注射相同的藥物,直至創(chuàng)面壞死愈合。術(shù)后第15天和25天過量水合氯醛處死大鼠,取背部壞死皮瓣標(biāo)本,進(jìn)行組織化學(xué)Masson染色和免疫組化染色分析創(chuàng)緣上皮及上皮下組織的生長情況,上皮的生長厚度,并取血清進(jìn)行ELISA檢測。 結(jié)果 1.酶切鑒定結(jié)果與設(shè)計(jì)和文獻(xiàn)相符,證明載體pAd-KGF構(gòu)建正確。pAd-KGF質(zhì)粒測序結(jié)果顯示起始位點(diǎn)：第185bp,ATG。終結(jié)位點(diǎn)：第679bp,TAA。全序列對比小鼠KGF基因序列,二者完全匹配,不存在突變。病毒轉(zhuǎn)染正常293細(xì)胞24小時,開始出現(xiàn)細(xì)胞變圓；熒光顯微鏡鏡下觀察,部分293細(xì)胞開始表達(dá)大量熒光蛋白。轉(zhuǎn)染第96小時后,所有293產(chǎn)生細(xì)胞病理效應(yīng),顯微鏡下觀察所有細(xì)胞呈圓球形葡萄樣改變,漂浮于培養(yǎng)液中,熒光顯微鏡鏡下觀察293細(xì)胞呈圓球形,表達(dá)大量熒光蛋白。重組腺病毒Ad-KGF和Ad在293細(xì)胞內(nèi)大量擴(kuò)增,并收集后。測得Ad-KGF病毒的效價滴度為2×1011PFU/ml,Ad病毒的效價滴度為3×1012PFU/ml。 2.pAd-KGF為構(gòu)建腺病毒質(zhì)粒。半定量PCR證明pAd-KG內(nèi)存在KGF表達(dá)基因。經(jīng)過基因重組后的腺病毒Ad-KGF具有感染正常細(xì)胞,于正常細(xì)胞內(nèi)表達(dá)KGF的能力。正常的293細(xì)胞不能表達(dá)KGF的mRNA,PA317細(xì)胞能分泌KGF,表達(dá)KGF mRNA。Ad-KGF感染293細(xì)胞后,細(xì)胞成功表達(dá)KGF mRNA,而在293細(xì)胞和293+空病毒Ad內(nèi)不能表達(dá)KGF mRNA。通過ELISA檢測培養(yǎng)液上清中外分泌的KGF含量。檢測證明Ad空病毒轉(zhuǎn)染細(xì)胞后48小時,上清液中不分泌KGF蛋白,而Ad-KGF轉(zhuǎn)染細(xì)胞的后的48小時,細(xì)胞培養(yǎng)上清中能夠檢測到大量的KGF表達(dá),且濃度隨著Ad-KGF的滴度增加而增加。通過劃痕實(shí)驗(yàn),證明在KGF作用下,HaCaT上皮細(xì)胞的遷移能力顯著增加。而對照組細(xì)胞開始出現(xiàn)凋亡現(xiàn)象。Transwell細(xì)胞遷移實(shí)驗(yàn)證明KGF能有效促進(jìn)HaCaT上皮細(xì)胞生長遷移,而對HUVEC血管內(nèi)皮細(xì)胞作用不明顯,對PA317成纖維細(xì)胞沒有明顯作用。 3.大鼠背部七條靜脈和一側(cè)有超過二十條動脈分布。手術(shù)后三天,大鼠的體重會稍有下降,然后穩(wěn)定上升。PBS組和空白組中壞死面積在9天內(nèi)達(dá)到最高峰。從9至47天中,壞死面積逐漸減少,在47天壞死幾乎完全愈合。組織病理檢查中,可見術(shù)后15天,壞死創(chuàng)口邊緣上皮細(xì)胞增生明顯,有少量纖維成分生成,創(chuàng)口愈合區(qū)有大量小脈管形成和大量不成熟的纖維成分形成。至第35天,上皮增生修復(fù)較前明顯弱,但較正常上皮,厚度仍明顯增大。 4.成功建立大鼠背部皮瓣壞死模型后,四組動物術(shù)后體重均有輕度下降,從第三天開始,體重逐步上升,其中以DXM組的體重上升最明顯。術(shù)后第五天,壞死成活區(qū)域分界清晰。術(shù)后第十天,壞死面積達(dá)到最大。隨著不同藥物的治療,壞死面積在各組間都明顯下降,且Ad-KGF組壞死面積縮小最為明顯。Masson染色顯示術(shù)后15天,四組壞死創(chuàng)口邊緣上皮都明顯增生,其中Ad-KGF組上皮細(xì)胞增生最多。術(shù)后35天,Ad-KGF組壞死傷口已經(jīng)愈合,而其它三組還有較大的壞死面積。術(shù)后15天和25天,上皮細(xì)胞厚度在Ad-KGF組增生最為明顯,術(shù)后35天上皮厚度與其它組間沒有大的差異。 免疫組織化學(xué)分析顯示,只有在Ad-KGF組的上皮和間充質(zhì)細(xì)胞中有強(qiáng)陽性的KGF表達(dá),而其它三組均為陰性。Anti-CD34免疫組織化學(xué)染色顯示四組的小血管生成數(shù)目相當(dāng) 5.PA317在EGF, VEGF, VEGF+EGF,或PA317作用下,沒有顯著性的生長差異。HUVEC對VEGF表現(xiàn)出明顯生長加速,在EGF和VEGF聯(lián)合作用下,生長更加明顯,而與PA317共培養(yǎng)時,也有少量生長改變。HaCaT細(xì)胞在EGF和VEGF+EGF聯(lián)合作用下都是表現(xiàn)出生長遷移能力提高。建立動物模型后,分組如下:PBS, EGF, VEGF, EGF+VEGF。四組動物術(shù)后體重均有輕度下降,從第三天開始,體重逐步上升,其中以EGF組的體重上升最少。隨著不同藥物的治療,壞死面積在各組間都明顯下降,且VEGF+EGF組壞死面積縮小最為明顯。 術(shù)后15天和25天,Anti-CD34免疫組織化學(xué)染色顯示EGF,VEGF和VEGF+EGF組的小微血管生成數(shù)目相當(dāng),都顯著高于PBS組。 結(jié)論 1. pAd-KGF經(jīng)過酶切鑒定和測序鑒定具有KGF完全匹配的基因組。成功構(gòu)建Ad.KGF和Ad,且具有感染細(xì)胞的能力。Ad.KGF和Ad經(jīng)過擴(kuò)增純化的滴度為2×1011PFU/ml和3×1012PFU/ml,可以滿足細(xì)胞及大鼠皮瓣實(shí)驗(yàn)的要求。 2. Ad-KGF和Ad可以高效轉(zhuǎn)染PA317、HUVEC和HaCaT細(xì)胞,并在轉(zhuǎn)染后高效表達(dá)活性KGF、KGF可有效促進(jìn)HaCaT細(xì)胞生長,而對PA317、HUVEC細(xì)胞作用不明顯。 3.本實(shí)驗(yàn)在確定大鼠背部血供系統(tǒng)基礎(chǔ)上,設(shè)計(jì)形成一套簡便且易于推廣的手術(shù)和皮瓣壞死模型流程,成功建立了穩(wěn)定的大鼠背部皮瓣壞死傷口模型,可以進(jìn)行皮瓣壞死為目的基因或藥物實(shí)驗(yàn)研究。 4.使用高表達(dá)KGF的腺病毒重組局部轉(zhuǎn)染大鼠背部皮瓣壞死傷口,能有效促進(jìn)壞死傷口的愈合。 5. EGF-VEGF聯(lián)合應(yīng)用能促進(jìn)改良大鼠背部壞死創(chuàng)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R782.2
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本文編號：2035191