Lgr5通过VEGF介导的血管生成调节结直肠癌的转移

发布时间：2018-05-09 04:15

本文选题：结直肠癌 + 肿瘤转移　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景结直肠癌已成为我国发病率增长最快的恶性肿瘤之一,然而在结直肠癌相关性死亡中,大部分为转移癌所致。近年来越来越多的研究聚焦于肿瘤转移,并不断探索肿瘤转移的机制,旨在减少肿瘤转移的发生,降低转移癌的死亡率。肿瘤血管生成是肿瘤生长以及肿瘤转移过程中非常重要的环节,VEGF是促进血管生成的重要因子,在肿瘤血管生成中也具有重要作用。目前有实验通过抑制VEGF的生成来抑制肿瘤血管形成,从而抑制肿瘤,但是对于结直肠癌的转移抑制作用不明显。因此,有必要进一步研究VEGF在结直肠癌形成以及转移过程中的机制。近年来,研究发现Lgr5为小肠、结肠以及毛囊等组织的干细胞特异性分子标记物,并且在肝细胞癌、结肠癌、卵巢癌、基底细胞癌以及胃癌等实体肿瘤中高表达。Lgr5是Wnt信号通路的靶基因,并且可以反馈性地调节Wnt信号通路的活性,从而调控细胞的生长、运动和分化。越来越多的研究证实,Lgr5可能也为结直肠癌的肿瘤干细胞标记物,可以与其他肿瘤干细胞标记物一同用于结直肠癌肿瘤干细胞的筛选。根据“肿瘤干细胞学说”,有数据表明,结直肠癌可能来源于隐窝基底部Lgr5阳性的干细胞。这就证明Lgr5可能对于结直肠癌的发生发展具有重要作用。然而,目前关于Lgr5在肿瘤形成、发展以及转移过程中的作用及其机制的研究较少。我们课题组前期证实在结直肠癌细胞中敲低Lgr5的表达后可以抑制肿瘤的血管生成。研究目的为了避免单方面敲低Lgr5表达引起实验结果的偶然性以及进一步探讨Lgr5在结直肠癌及其转移癌中的作用,Lgr5对VEGF的影响及其机制,为以Lgr5为靶点的结直肠癌治疗提供理论依据。研究方法 1.构建Lgr5过表达质粒并建立稳定细胞株根据完整的人Lgr5(NM_003667)基因序列信息设计其编码区扩增引物,引物序列见表一,引物扩增总长度为2745bps。将其克隆到pcDNA3.1(+)真核表达载体中,经检测序列正确。利用Lipofectamine2000将pcDNA3.1(+)-Lgr5质粒、pcDNA3.1(+)空载体质粒瞬时转染入SW480细胞,48小时后,以1000ug/mlG418进行连续筛选,约2-3周挑克隆并扩大培养,以600ug/mlG418继续维持。 2. Western blotting 提取细胞的总蛋白,用BCA蛋白质定量试剂盒检测蛋白浓度,蛋白质样品上样量为20μg。电泳后将蛋白质电转至PVDF膜,5%脱脂牛奶封闭,4℃孵育一抗过夜,一抗稀释比例为(Lgr51:500, VEGF1:200, GAPDH1:1000,β-actin 1：1000),次日二抗孵育1小时,ECL化学发光法显影。 3.实时荧光定量PCR (qRT-PCR) 提取细胞、组织总RNA并测定RNA浓度,用M-MLV逆转录酶合成cDNA第一链,使用Takara的SYBR Premix Ex Taq试剂盒进行实时荧光定量PCR,引物序列见表一。 4.免疫组织化学(SP法) 收集南方医院结直肠原发癌标本58例,结直肠癌转移癌标本8例,组织经10%福尔马林固定,石蜡包埋,标本切成4μm薄片,65℃烤箱烤片约3小时,依次脱蜡水化,以0.01M枸橼酸钠缓冲液(pH6.0)高温微波修复15mmin,利用迈新公司UltraSensitiveS-P超敏试剂盒,一抗4℃冰箱孵育过夜(Lgr51:50、CD311:50),经DAB显色剂显色,苏木素复染,脱水透明,封片风干,显微镜下观察并拍片,两个病理科医生分别独立阅片并评分。 5.HE染色组织经10%福尔马林固定,石蜡包埋,标本切成4μ.m薄片,65℃烤箱烤片约30分钟,依次脱蜡水化,苏木素染色2-5分钟,1%盐酸酒精分化,流水冲洗返蓝15分钟,伊红染色数秒至数分钟,脱水透明,封片晾干,显微镜下观察并拍片。 6. Transwell小室迁移与侵袭实验将100u1浓度为以1x106/ml的细胞悬液加于上室,然后将500u1含10%FBS的培养基加于下室,培养24小时后,取出小室,4%多聚甲醛将细胞固定30分钟,结晶紫中染色10分钟,用棉签擦掉上室残留细胞,显微镜下随机选取5个视野进行细胞计数。侵袭实验在上室铺有一层基质胶,其余方法及操作均同迁移实验。 7.小管形成实验质粒瞬时转染后48小时收集培养基,经孔径为0.22μmn的滤器分别过滤,收集为条件培养基。基质胶加入96孔板中,每孔加入50ul,37℃温度下交联1小时使胶固化。每孔加入50u1浓度为2×105/ml的HMVEC细胞,并且加入相应的条件培养基。于0h、2h、4h、8h在倒置显微镜下观察HMVEC细胞形成小管样结构的情况,并拍照,计数视野中小管样网状结构中的闭合环形小管结构的个数。 8. Elisa实验根据人血管内皮生长因子(VEGF) Elisa试剂盒(武汉华美生物工程有限公司)的操作说明,检测重组细胞培养基中VEGF的浓度。 9. AOM/DSS模型的建立选取5-6周balb/c小鼠,随机分成2组,每组10只。实验组用终浓度为1.25mg/ml的AOM按照12.5mg/kg的剂量腹腔注射一次(10ul/g),对照组以腹腔注射等剂量生理盐水。对照组不做特殊处理,实验组进行如下处理：第1周正常饮水；第2周饮水含2.5%DSS,第3、4周连续2周正常饮水,此为一个循环；第5-10周,重复第2、3、4周处理2次,即总共三个循环。继续饲养至17周,处死老鼠并取结肠组织,观察其瘤体情况,将结肠以10%福尔马林固定,石蜡包埋,行HE染色。提取结肠组织RNA,并进一步行实时荧光定量PCR检测Lgr5以及VEGF的表达量。 10.裸鼠成瘤与肝转移实验裸鼠成瘤实验选取5-6周Balb/c裸鼠,随机分成2组,每组7只,饲养等级为SPF级。分别注射SW620Lgr5shRNA细胞、SW620NC细胞至裸鼠背侧,调整细胞浓度为1×107/ml,注射200ul,注射细胞数为2×106个。观察裸鼠的成瘤情况,每隔3天称量裸鼠的体重,测量肿瘤的长径(L)以及宽径(W),以公式L×w2/2计算出瘤体的体积。4周后,裸鼠安乐死,并取下瘤体,经10%福尔马林固定石蜡包埋,供后续HE染色。裸鼠结直肠癌肝转移实验前期细胞准备与裸鼠同成瘤实验,以1%戊巴比妥钠6-8ul/g腹腔注射麻醉裸鼠,在裸鼠左侧背部取长约1cm小口,暴露脾脏,沿着脾门上缘从脾脏头侧进针向脾脏尾部注射,将100ul浓度为1×107/ml的细胞缓慢注入脾被膜下,注射成功后,清理腹腔,逐层关腹并逐层缝合,并以纱布包扎。观察裸鼠的生长状态,每隔3天称量裸鼠的体重。3周后,处死裸鼠,并取下脾脏、肝脏、肺脏等组织,经10%福尔马林固定石蜡包埋,供后续HE染色。结果 1.免疫组化初步结果显示,在结直肠原发癌以及转移癌中,存在Lgr5表达越高,微血管密度(MVD)越高的趋势对结直肠原发癌、淋巴结转移癌、肝转移癌组织标本进行免疫组织化学染色,检测Lgr5以及血管内皮标记物CD31的表达,发现Lgr5的表达在淋巴结转移癌与肝转移癌中表达更高,并且CD31的表达同样也升高,即微血管密度升高。免疫组化初步结果显示,在结直肠原发癌以及转移癌中,存在Lgr5表达越高,微血管密度(MVD)越高的趋势,然而这种趋势以及二者的相关性还需要通过进一步加大样本量,获得统计学数据加以证明。 2.成功构建高表达Lgr5的稳定细胞株转染Lgr5重组过表达质粒pcDNA3.1(+)-Lgr5及pcDNA3.1(+)空载体质粒进入SW480并采用G418进行筛选,分别采用western blotting与实时荧光定量PCR技术检测Lgr5在SW480Lgr5与SW480vector中蛋白质以及mRNA的表达水平。结果发现,与SW480vector相比,Lgr5的蛋白质表达水平在SW480Lgr5细胞中明显升高；Lgr5的mRNA表达水平在SW480Lgr5细胞中同样明显升高(t=-17.499,p0.05)。 3.Lgr5对肿瘤细胞迁移与侵袭能力的影响在迁移与侵袭实验中,与SW480vector细胞组相比,SW480Lgr5细胞组迁移与侵袭的细胞数明显增多(迁移：t=4.291,p0.05；侵袭：t=3.127,p0.05)；与SW620NC细胞组相比,SW620Lgr5shRNA细胞组迁移与侵袭的细胞数明显减少(迁移：t=6.557,p0.01；侵袭：t=2.297, p0.05)。Transwell小室迁移与侵袭实验表明,升高Lgr5表达后肿瘤细胞的迁移与侵袭能力明显增强,敲除Lgr5表达后肿瘤细胞的迁移与侵袭能力明显减弱。 4.Lgr5对内皮细胞小管形成能力的影响用内皮细胞在Matrigel中形成闭合环形小管的数目表示内皮细胞小管形成的能力。与SW480vector组相比,SW480Lgr5组小管样结构明显增多(t=8.617,p0.05)；与SW620NC组相比,SW620Lgr5shRNA组小管样结构数明显减少(t=5.670,p0.05)。 5.Lgr5对结直肠癌细胞内血管内皮生长因子的影响通过Western blotting发现,与SW480vector细胞相比,SW480Lgr5细胞中VEGF的蛋白含量明显增多,与SW620NC细胞相比,SW620Lgr5shRNA细胞VEGF的蛋白含量明显减少。通过Elisa发现,与SW480vector组相比,SW480Lgr5组中分泌VEGF的量明显增多(t=12.58,p0.01),与SW620NC组相比,SW620Lgr5shRNA组分泌VEGF的蛋白含量明显减少(t=8.104,p0.01)。 6. AOM/DSS模型中,肿瘤组织Lgr5与VEGF的mRNA表达明显增高成功建立AOM/DSS模型,提取建模成功后的小鼠结肠组织RNA,行qRT-PCR显示,与对照组相比,实验组中Lgr5以及VEGF的mRNA的表达量明显升高(Lgr5:t=14.30, p0.01; VEGF:t=9.213, p0.01)。 7.敲低Lgr5的表达后,降低结直肠癌细胞在裸鼠体内的成瘤以及肝转移能力在成瘤实验中,NC组的瘤体体积明显大于Lgr5shRNA组,并且这种显著差异在第3周开始体现出来。在裸鼠结肠癌肝转移实验中,与NC组相比,Lgr5shRNA组肝脏内癌结节数目明显较少(t=-4.386,p0.05)。结论通过以上实验结果,我们可以得出以下结论： 1.在体外,Lgr5可以促进结直肠癌细胞的迁移与侵袭能力； 2.在体内,Lgr5可以促进结直肠癌的成瘤以及肝转移； 3.Lgr5可以促进内皮细胞的血管生成,可以促进结直肠癌中VEGF的表达；我们通过功能性获得与失去实验、体内外实验,发现在结直肠癌中,Lgr5可能是通过促进VEGF的表达,从而促进血管生成,最终促进肿瘤形成与转移。因而我们的研究可以为以Lgr5为靶点的治疗策略提供理论依据。
[Abstract]:Research background
Colorectal cancer has become one of the fastest growing malignant tumors in China. However, in colorectal cancer related deaths, most of them are caused by metastatic cancer. In recent years, more and more studies focus on tumor metastasis and explore the mechanism of tumor metastasis to reduce the occurrence of tumor metastasis and reduce the mortality of metastatic cancer. Guan Shengcheng is a very important link in the process of tumor growth and tumor metastasis. VEGF is an important factor promoting angiogenesis and also plays an important role in angiogenesis. At present, there are experiments to inhibit the formation of VEGF to inhibit tumor angiogenesis and inhibit tumor, but the inhibition of metastasis of colorectal cancer is unknown. Therefore, it is necessary to further study the mechanism of VEGF in the process of colorectal cancer formation and metastasis.
In recent years, Lgr5 has been found to be a specific marker of stem cell specific molecules in small intestine, colon, and hair follicle, and high expression of.Lgr5 is the target gene of Wnt signaling pathway in hepatocellular carcinoma, colon cancer, ovarian cancer, basal cell carcinoma and gastric cancer, and can regulate the activity of Wnt signaling pathway by feedback. More and more studies have shown that Lgr5 may also be a marker for cancer stem cells in colorectal cancer and can be used with other tumor stem cell markers for screening cancer stem cells in colorectal cancer. According to the "tumor stem cell theory", the data suggest that colorectal cancer may originate from the basement of the crypts of Lgr. 5 positive stem cells. This suggests that Lgr5 may play an important role in the development of colorectal cancer. However, there are few studies on the role and mechanism of Lgr5 in the process of tumor formation, development and metastasis. Our group has previously confirmed that the tumor's blood vessels could be suppressed after the low Lgr5 expression in colorectal cancer cells Generate.
research objective
In order to avoid the incidental results of Lgr5 expression, and to further explore the role of Lgr5 in colorectal cancer and its metastatic carcinoma, the effect of Lgr5 on VEGF and its mechanism will provide a theoretical basis for the treatment of colorectal cancer with Lgr5 as the target.
research method
1. construction of Lgr5 overexpression plasmid and establishment of stable cell line
The amplified primers were designed according to the sequence information of the complete human Lgr5 (NM_003667) gene sequence. The primer sequence was shown in Table 1. The primer amplification length was 2745bps. and was cloned into the pcDNA3.1 (+) eukaryotic expression vector. The sequence was correct. PcDNA3.1 (+) -Lgr5 plasmid and pcDNA3.1 (+) empty body particles were transfected into SW4 instantaneously by Lipofectamine2000. 80 cells were continuously screened by 1000ug/mlG418 for 48 hours, and then cloned and expanded for about 2-3 weeks to maintain 600ug/mlG418.
2. Western blotting
The total protein of the cell was extracted and the protein concentration was detected by the BCA protein quantitative kit. After the sample sample was 20 u g. electrophoresis, the protein was electrically transferred to the PVDF membrane, the 5% skimmed milk was closed, and a night resistance was incubated at 4. The first anti dilution ratio was (Lgr51:500, VEGF1:200, GAPDH1:1000, beta -actin).
1:1000), two days after incubation for 1 hours, ECL was developed by chemiluminescence.
3. real time fluorescence quantitative PCR (qRT-PCR)
The cells were extracted, the total RNA was organized and the concentration of RNA was measured. The first chain of cDNA was synthesized by the M-MLV reverse transcriptase. The real-time fluorescent quantitative PCR was carried out by the SYBR Premix Ex Taq kit of Takara. The primer sequence was shown in Table 1.
4. immuno histochemistry (SP method)
58 specimens of primary colorectal cancer in the southern hospital and 8 cases of metastatic carcinoma of colorectal cancer were collected, the tissues were fixed by 10% formalin, paraffin embedded, the specimens were cut into 4 micron m slices, 65 Centigrade oven roasted slices for 3 hours, and then dewaxing and hydrating, and using 0.01M sodium citrate buffer (pH6.0) to repair 15mmin with high temperature microwave, and the UltraSensitiveS-P hypersensitivity test of Maoxin company was used. The agent box, one anti 4 C refrigerator incubated for night (Lgr51:50, CD311:50), was coloured by DAB coloring agent, restained with hematoxylin, dehydrated transparent, sealed by wind, observed under microscope and filming. Two pathologist independently read the film and score.
5.HE staining
The tissue was fixed by 10% formalin, paraffin was embedded, the specimens were cut into 4 mu.M slices, and the roast slices of 65 Centigrade oven were about 30 minutes, and then dewaxing and hydrating in turn. The hematoxylin was stained for 2-5 minutes, 1% hydrochloric acid alcohol was differentiated, the water was washed to blue for 15 minutes, the eosin was stained for several seconds to several minutes, the dehydration was transparent, the seal was dried, under microscope observation and flapping.
6. Transwell compartment migration and invasion experiment
The concentration of 100u1 was added in the cell suspension with 1x106/ml in the upper chamber, then the medium of 500u1 containing 10%FBS was added to the lower chamber. After 24 hours culture, the cell was removed and 4% polyformaldehyde was fixed for 30 minutes, and the crystal violet was stained for 10 minutes. The residual cells in the upper chamber were wiped out with the cotton swab. The cells were randomly selected under the microscope microscope. The cells were counted and the cells were counted. The invasion experiment was carried out. In the upper chamber there is a layer of matrix adhesive, the rest of the methods and operations are in the same migration test.
7. tubule formation experiment
The medium was collected 48 hours after the plasmid transfection. The medium was filtered by the filter with the pore size of 0.22 Mn respectively. The medium was collected as the conditioned medium. The matrix gum was added to the 96 hole plate, each hole was added to 50ul, and the glue was cured for 1 hours at 37 C. The HMVEC cells with the concentration of 2 x 105/ml were added to each hole, and the corresponding conditioned medium was added. 0h, 2h, 4h, 8h were at 0h. The microtubule like structure of HMVEC cells was observed under the inverted microscope, and the number of closed tubular structures in the meso tube reticular structure was counted.
8. Elisa experiment
According to the operation instructions of human vascular endothelial growth factor (VEGF) Elisa Kit (Wuhan Huamei Biological Engineering Co., Ltd.), the concentration of VEGF in the recombinant cell culture medium was detected.
The establishment of 9. AOM/DSS model
5-6 weeks of balb/c mice were randomly divided into 2 groups, each group had 10 rats in each group. The experimental group was injected with AOM of the final concentration of 1.25mg/ml according to the dose of 12.5mg/kg (10ul/g), and the control group was injected with equal dose of saline in the abdominal cavity. The control group did not do special treatment. The experimental group was treated as the following: first weeks of normal drinking water; second weeks of drinking water containing 2.5%DSS, third, 4 weeks of continuous 2 weeks of normal drinking water, this is a cycle, this is a cycle of 5-10 weeks, repeat week 2,3,4 treatment 2 times, that is, a total of three cycles. Continue to feed to 17 weeks, kill the mice and take the colon tissue, observe the tumor body condition, the colon is fixed with 10% formalin, paraffin embedded, HE color. Extract the colon tissue RNA, and walk in real-time real-time quantitative PC fluorescence quantitative PC R detected the expression of Lgr5 and VEGF.
10. experimental tumor and liver metastases in nude mice
Tumor formation experiment in nude mice
5-6 weeks of Balb/c nude mice were randomly divided into 2 groups, 7 rats in each group, and the feeding grade was SPF grade. SW620Lgr5shRNA cells were injected respectively, SW620NC cells to the dorsal side of nude mice. The cell concentration was 1 x 107/ml, 200ul was injected, and the number of injected cells was 2 x 106. The tumor formation of nude mice was observed, the length of the nude mice was weighed every 3 days and the length of tumor (L) was measured and measured. A wide diameter (W) was used to calculate the volume of the tumor by formula L * w2/2. After.4 weeks, the nude mice were euthanized and the tumor was removed. The tumor was then embedded in 10% formalin fixed paraffin for subsequent HE staining.
Experimental liver metastases in nude mice with colorectal cancer
The early cell preparation was prepared with the nude mice, with 1% pentobarbital sodium 6-8ul/g injected into the nude mice by intraperitoneal injection of nude mice. The spleen was taken on the left back of the nude mice and the spleen was exposed. The spleen was injected along the upper margin of the spleen from the spleen to the tail of the spleen. The cells with 100ul concentration of 1 x 107/ml were injected into the splenic membrane slowly, and the abdominal cavity was cleaned after the injection was successful. The growth state of nude mice was observed and the nude mice were weighed every 3 days after.3 weeks. The nude mice were killed and the spleen, liver, lung and other tissues were removed, and 10% formalin fixed paraffin was embedded for subsequent HE staining.
Result
1. immunohistochemical results showed that the higher the expression of Lgr5 and the higher the microvessel density (MVD) were in colorectal cancer and metastatic cancer.
The expression of Lgr5 and vascular endothelial marker CD31 was detected by immunohistochemical staining in the specimens of primary colorectal cancer, lymph node metastasis and liver metastasis. It was found that the expression of Lgr5 was higher in lymph node metastasis and liver metastasis, and the expression of CD31 was also elevated, that is, the increase of microvascular density. The higher the expression of Lgr5 and the higher the microvessel density (MVD) in colorectal primary and metastatic cancers, however, the trend and the correlation between the two also need to be obtained by further increasing the sample size to obtain statistical data.
2. the successful construction of a stable cell line with high expression of Lgr5
The transfected Lgr5 recombinant overexpressed plasmid pcDNA3.1 (+) -Lgr5 and pcDNA3.1 (+) empty body particles entered SW480 and screened by G418. Western blotting and real-time fluorescent quantitative PCR technique were used to detect the protein and expression level of Lgr5 in SW480Lgr5 and SW480vector. The level of SW480Lgr5 increased significantly in Lgr5 cells, and the expression level of mRNA in SW480Lgr5 cells was also significantly increased (t=-17.499, P0.05).
Effects of 3.Lgr5 on migration and invasion of tumor cells
In the migration and invasion experiments, the number of migrating and invading cells in the SW480Lgr5 cell group increased significantly compared with the SW480vector cell group (migration: t=4.291, P0.05; invasion: t=3.127, P0.05); compared with the SW620NC cell group, the number of cells migrated and invaded by the SW620Lgr5shRNA cell group decreased significantly (migration: t=6.557, P0.01; invasion: t=2.297, P0.05).Transwell cell migration and invasion experiments showed that the migration and invasion ability of tumor cells increased obviously after the increase of Lgr5 expression, and the migration and invasion ability of tumor cells decreased significantly after knocking out Lgr5 expression.
Effect of 4.Lgr5 on endothelial cell tubule formation
The number of closed loop tubules formed by endothelial cells in Matrigel showed the ability to form endothelial cells. Compared with the SW480vector group, the tubule like structure in the SW480Lgr5 group increased significantly (t=8.617, P0.05). Compared with the SW620NC group, the number of tubules in the SW620Lgr5shRNA group decreased significantly (t=5.670, P0.05).
Effect of 5.Lgr5 on vascular endothelial growth factor in colorectal cancer cells
Compared with SW480vector cells, the protein content of VEGF increased significantly in SW480Lgr5 cells compared with SW480vector cells. Compared with SW620NC cells, the protein content of VEGF in SW620Lgr5shRNA cells decreased significantly. Compared with the SW480vector group, the amount of VEGF in SW480Lgr5 group increased significantly compared with the SW480vector group. Compared with group SW620Lgr5shRNA, the protein content of VEGF secreted significantly decreased (t=8.104, P0.01).
In the 6. AOM/DSS model, the expression of Lgr5 and VEGF mRNA in tumor tissues increased significantly.
The AOM/DSS model was successfully established to extract the RNA of mouse colon tissue after successful modeling, and qRT-PCR was shown. Compared with the control group, the expression of Lgr5 and VEGF in the experimental group increased significantly (Lgr5:t=14.30, P0.01; VEGF:t=9.213, P0.01).
7. knocking down the expression of Lgr5 decreases the tumorigenesis and liver metastasis of colorectal cancer cells in nude mice.
In the tumor formation experiment, the volume of the tumor body in the NC group was significantly larger than that in the Lgr5shRNA group, and the significant difference was found at third weeks. In the liver metastasis test of nude mice, the number of tumor nodules in the Lgr5shRNA group was significantly less than that in the NC group (t=-4.386, P0.05).
conclusion
From the above results, we can draw the following conclusions:
1. in vitro, Lgr5 can promote the migration and invasion of colorectal cancer cells.
2. in vivo, Lgr5 can promote tumor formation and liver metastasis of colorectal cancer.
3.Lgr5 can promote the angiogenesis of endothelial cells and promote the expression of VEGF in colorectal cancer.
Through functional acquisition and loss of experiments and in vitro and in vivo experiments, we found that in colorectal cancer, Lgr5 may promote angiogenesis by promoting the expression of VEGF, and ultimately promote tumor formation and metastasis. Therefore, our research can provide a theoretical basis for the therapeutic strategy targeting Lgr5.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R735.34

【相似文献】