密码子优化的RHDV2双VP60基因重组杆状病毒构建及表达蛋白免疫原性分析

[目的]试验旨在优化兔出血症病毒2型(Rabbit hemorrhagic disease virus 2,RHDV2)病毒样颗粒(virus-like particle, VLP)疫苗的制备策略,探究RHDV2 VLP疫苗对家兔的免疫原性,为低成本、高产量RHDV2新型疫苗研发提供新思路。[方法]根据昆虫细胞的密码子偏好性优化合成RHDV2 VP60全基因,将双VP60基因插入真核载体pFastBacTMDual,转化携带Bacmid质粒的大肠杆菌DH10Bac感受态细胞,构建含双VP60基因的重组杆粒Bacmid-VP60-VP60,转染Sf9昆虫细胞,通过Western blotting、间接免疫荧光试验(IFA)及透射电镜对重组杆状病毒Bacmid-VP60-VP60进行表达验证;将优化策略制备的重组蛋白抗原与氢氧化铝佐剂按照9∶1比例制备VLP灭活疫苗,通过安全性检验、最小免疫剂量、免疫持续期等评估优化策略制备的RHDV2 VLP疫苗的保护效果。[结果]试验成功构建重组杆粒Bacmid-VP60-VP60。Western blotting鉴定结果显示,重组杆状病毒转染Sf9细胞表达出大小约60 ku的RHDV2 VP60蛋白。IFA鉴定结果显示,感染重组杆状病毒的Sf9细胞产生了大量的黄绿色荧光,表明重组杆状病毒在Sf9细胞中大量表达VP60蛋白。透射电镜观察结果显示,VP60蛋白折叠成VLP,大小为40 nm左右,呈现球形结构,表面光滑。优化策略制备的RHDV2 VLP疫苗对家兔具有良好的安全性和免疫原性,最小免疫剂量为0.5 mL/只,免疫持续期可达210 d以上。[结论]试验构建了含有密码子优化的双VP60基因的重组杆状病毒,并在昆虫细胞中成功表达RHDV2 VP60蛋白,该蛋白制备的VLP疫苗对家兔具有良好的免疫原性。

超声引导下组织块接种制作兔肾VX_2瘤模型及传代保存

目的 探讨术中组织块包埋和超声引导下穿刺组织块接种制作兔肾VX2 瘤模型的方法 ,并对这两种方法进行比较。方法 采用剪切法制备兔VX2 瘤组织块悬液 ,应用超声引导下经皮组织块推注法建立 5 8只兔肾VX2 肿瘤模型 ,应用术中组织块包埋法建立 8只兔肾VX2 肿瘤模型 ,应用自然组织谐波技术观察肿瘤生长 ,并观察浅表淋巴结转移及动物的一般情况 ;3周内自然死亡者立即行尸体解剖 ,未死者种植后 3周处死行尸体解剖 ,获得兔肾VX2 肿瘤标本 ,进行HE切片染色。结果 两种方法建立的兔肾VX2 瘤模型组织学表现与传代种兔相同 ;术中包埋组种植成功率 5 0 % ,超声引导下穿刺组种植成功率 96.5 5 % ;术中包埋组腹壁种植率 2 5 % ,超声引导下穿刺组腹壁种植率 5 .17%。结论 超声引导下穿刺法建立兔肾VX2 肿瘤模型方法简便 ,成功率高 。

兔VX_2肝癌模型的建立及超声评价

目的 探讨不同方法制作兔VX2 移植性肝癌模型的特点 ,评价超声检查在监测VX2 肝癌中的价值。方法 将VX2 细胞滤液、VX2 瘤株小块及VX2 瘤株小块 +明胶海绵分别注射或接种于 2 8只新西兰大白兔的肝脏内 ,一周后不同时间行超声检查。结果 A组滤液注射法 :平均成瘤时间约 2 1天 ,肝的原位成瘤率为 75 %。B组包块埋植法 :平均成瘤时间14天 ,肝的原位成瘤率为 83 %。C组包块埋植 +明胶海绵法 :平均成瘤时间 14天 ,肝的原位成瘤率 88%。三组成瘤率统计学无显著差异 ( χ2 =1.118,P >0 .0 5 )。肝脏内的移植瘤在超声上均表现为低回声伴声晕的肿块 ,彩色多普勒血流成像表现为周边血供丰富 ,中央血供稀少。结论 三种方法均能制成肝癌模型 ,超声检查是一种监测肝癌模型的有效手段。

组织块悬液注射法制作兔VX_2乳腺癌模型

目的 :建立稳定的兔VX2 乳腺癌模型 ,选择最佳的模型制作方法。方法 :3 0只兔随机分为 3组 ,每组10只 ,分别采用乳腺内瘤细胞液注射法、组织块悬液注射法、组织块种植法制作兔VX2 乳腺癌模型 ,比较各组的种植成活率、肿瘤生长率、荷瘤兔存活时间 ,并观察肿瘤的生物学形态。结果 :组织块悬液注射法操作简便 ,成瘤率高 ,肿瘤增殖旺盛 ,生物学特征符合乳腺鳞状细胞癌。结论 :成功建立了兔VX2 乳腺癌模型 。

肠艾美耳球虫重组微线蛋白2对家兔免疫保护效果评价

本研究旨在评价肠艾美耳球虫重组微线蛋白2(rEiMIC2)对兔的免疫保护效果,为肠艾美耳球虫重组亚单位疫苗的研究提供参考。在肠艾美耳球虫转录组数据中筛选出EiMIC2基因,进行克隆、原核表达和蛋白纯化,并采用免疫印迹检测重组蛋白的免疫反应性。将35日龄无球虫新西兰幼兔32只随机分为4组(未免疫未攻虫组、未免疫攻虫组、pET-32a(+)载体组和rEiMIC2免疫组),每组8只,未免疫未攻虫组和未免疫攻虫组每只颈部皮下注射1 mL无菌PBS,pET-32a(+)载体组和rEiMIC2免疫组每只分别接种100μg pET-32a(+)载体蛋白、rEiMIC2,首免后2周进行二免。二免14 d后,除去未免疫未攻虫组,其余3组每只新西兰兔口服接种5×10^(4)个肠艾美耳球虫孢子化卵囊;攻虫14 d后安乐死所有新西兰兔。重组蛋白免疫兔后通过观察临床症状、肠道病变、测定兔的相对增重率、卵囊减少率、抗球虫指数(ACI)值和料肉比以及检测血清中特异性IgG、IL-2、IL-4、IL-10、IFN-γ综合评价rEiMIC2的免疫保护效果。结果显示,EiMIC2基因开放阅读框长度为1 605 bp,编码的蛋白分子质量约为57.68 ku。免疫印迹显示rEiMIC2能够被阳性血清所识别,表明其具有良好的免疫反应性。免疫保护试验显示:攻虫后各组幼兔陆续出现精神沉郁和腹泻的症状,未免疫攻虫组和rEiMIC2免疫组各有1只兔死亡;rEiMIC2免疫组相对增重率为73.97%,卵囊减少率为69.98%,ACI值为136.97,料肉比为3.98∶1,与未免疫攻虫组差异显著(P<0.05);rEiMIC2免疫组肠道病变与未免疫攻虫组相比较轻,血清中特异性IgG水平显著高于未免疫攻虫组(P<0.05)。综上,rEiMIC2免疫兔后,能有效减少卵囊排出和增重损失,减轻肠道损伤,具有一定的保护效果。

VX_2种植瘤^(99m)Tc-HL91 SPECT和^(18)F-FDG PET/CT显像的对比研究

目的:用荷瘤兔模型研究乏氧显像剂(4,9diaza3,3,10,10tetramethyldodecan2,11dionedioxime,99mTcHL91)和18F氟代脱氧葡萄糖(18Ffluorodeoxyglucose,18FFDG)在肿瘤乏氧诊断中的应用价值。方法:1)体外试验,分别观察在常氧及乏氧状态下对VX2细胞摄取99mTcHL91和18FFDG的影响。2)体内试验,分别于耳缘静脉注入99mTcHL91后3h和18FFDG后1h,采集荷瘤兔的图象,随后,处死荷瘤兔取脑、肺、心、肝、脾、肾、坏死和非坏死肿瘤等标本称质量,测定放射性计数。肿瘤组织切片行HE染色,观察其特点。结果:1)乏氧与常氧状态下比较能引起VX2细胞对99mTcHL91和18FFDG的摄取并随时间延长而增加,P<0.05。2)荷瘤兔肿瘤显像清晰,肿瘤未坏死部位的放射性分别是坏死部位的4倍(99mTcHL91)和3.5倍(18FFDG)。HE染色显示瘤内有大量的坏死组织。结论:乏氧能引起VX2细胞对99mTcHL91和18FFDG的摄取增加;VX2荷瘤兔的瘤体在99mTcHL91和18FFDG显像上均为清晰的阳性病灶,坏死肿瘤组织的放射性低于非坏死肿瘤组织;两者均不能将乏氧的部位明确地显示出来。

二维超声技术检测VX_2肿瘤所致兔上腔静脉阻塞

目的 :研究实验兔VX2 肿瘤性上腔静脉 (SVC)阻塞二维超声图像特征 .方法 :使用美国产Sequoia 5 12彩色电脑声像仪 ,在超声显像引导下将VX2 瘤组织悬液接种到 14只兔SVC前方软组织内 ,建立SVC阻塞的动物模型 12d后 ,每隔 2d经右锁骨上窝二维超声检测肿瘤、SVC形态及相关指标 .结果 :随着肿瘤生长对SVC产生压迫和 (或 )浸润 ,SVC管腔变形或管壁连续性中断 ,管腔内出现异常回声 ,管径局部或全程变窄 ,肿瘤直径与SVC狭窄程度呈明显负相关 (r =- 0 .933,P <0 .0 5 ) ;上腔静脉阻塞 4 2d模型超声检测结果与CT和尸检的结果比较无显著性差异 (P >0 .0 5 ) .结论 :兔VX2 肿瘤性SVC阻塞动物模型二维超声变化与肿瘤生长密切相关 ,与CT检查及尸体解剖测量结果一致性较好 .

RFA治疗对VX_2肝肿瘤兔免疫功能影响的实验研究

目的 :探讨RFA治疗对机体免疫功能的影响。方法 :采用开腹直视下行兔肝VX2 肿瘤RFA治疗 ,定期用MTT法检测兔外周血T淋巴细胞活性及分泌IL 2的能力和腹腔巨噬细胞的活性。结果 :RFA治疗组的荷瘤兔T淋巴细胞活性及分泌IL 2的能力和腹腔巨噬细胞的活性均较手术治疗组和自身治疗前有显著提高 (P <0 .0 5 )。结论 :RFA治疗可以提高机体的细胞免疫力 。

Establishment of Rabbit Liver VX2 Tumor Model Using Percutaneous Puncture Inoculation of Tumor Fragment Guided and Evaluated by Ultrasonography

The aim of the present study is to evaluate a method of establishing model of rabbit liver VX2 tumor using percutaneous puncture inoculation of tumor fragment guided by ultrasonography.VX2 tumor fragments were implanted into the liver of 13 New Zealand white rabbits flushed by 1 mL normal saline through percutaneous puncture needle guided by ultrasonography.Conventional ultrasonography and contrast-enhanced ultrasonography(CEUS)were performed 14 days after inoculation,and then the rabbits were sacrificed and pathologically examined.The success rate of inoculation was 100%.The average size of liver VX2 tumor was 1.7 cm×1.3 cm,CEUS of VX2 liver tumors showed the"rapid wash-in and wash-out"vascular pattern.There were significant differences between VX2 tumors and liver parenchyma in quantitative parameters of A,k and A×k(P<0.05),which meant that VX2 liver tumors were characterized by more blood flow volume and faster blood velocity than liver parenchyma.Tumor fragment flushed by normal saline into the liver through a needle may be a promising method for the induction of a hepatic tumor.And CEUS can be used for accurately assessing angiogenesis and blood perfusion of VX2 tumors.

Role of MR-DWI and MR-PWI in the radiotherapy of implanted pulmonary VX-2 carcinoma in rabbits

Objective： To detect the activity of tumor cells and tumor blood flow before and after the radiotherapy of implanted pulmonary VX-2 carcinoma in rabbit models by using magnetic resonance diffusion-weighted imaging（MR-DWI） and magnetic resonance perfusion weighted imaging（MR-PWI）, and to evaluate the effectiveness and safety of the radiotherapy based on the changes in the MR-DWI and MR-PWI parameters at different treatment stages.Methods： A total of 56 rabbit models with implanted pulmonary VX-2 carcinoma were established, and then equally divided into treatment group and control group. MR-DWI and MR-PWI were separately performed using a Philips Acheiva 1.5T MRI machine（Philips, Netherland）. MRI image processing was performed using special perfusion software and the WORKSPACE advanced workstation for MRI. MRDWI was applied for the observation of tumor signals and the measurement of apparent diffusion coefficient（ADC） values; whereas MR-PWI was used for the measurement of wash in rate（WIR）, wash out rate（WOR）, and maximum enhancement rate（MER）. The radiation treatment was performed using Siemens PRIMUS linear accelerator. In the treatment group, the radiotherapy was performed 21 days later on a once weekly dosage of 1,000 c Gy to yield a total dosage of 5,000 c Gy.Results： The ADC parameters in the region of interest on DWI were as follows： on the treatment day for the implanted pulmonary VX-2 carcinoma, the t values at the center and the edge of the lesions were 1.352 and 1.461 in the treatment group and control group（P〉0.05）. During weeks 0-1 after treatment, the t values at the center and the edge of the lesions were 1.336 and 1.137（P〉0.05）. During weeks 1-2, the t values were 1.731 and 1.736（P〈0.05）. During weeks 2-3, the t values were 1.742 and 1.749（P〈0.05）. During weeks 3-4, the t values were 2.050 and 2.127（P〈0.05）. During weeks 4-5, the t values were 2.764 and 2.985（P〈0.05）. The ADC values in the treatment group were significantly higher than in the control group. After the radiotherapy（5,000 c Gy）, the tumors remarkably shrank, along with low signal on DWI, decreased signal on ADC map, and remarkably increased ADC values. As shown on PWI, on the treatment day for the implanted pulmonary VX-2 carcinoma, the t values of the WIR, WOR, and MER at the center of the lesions were 1.05, 1.31, and 1.33 in the treatment group and control group（P〉0.05）; in addition, the t values of the WIR, WOR, and MER at the edge of the lesions were 1.35, 1.07, and 1.51（P〉0.05）. During weeks 0-1 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 1.821, 1.856, and 1.931（P〈0.05）; in addition, the t values of the WIR, WOR, and MER at the edge of the lesions were 1.799, 2.016, and 2.137（P〈0.05）. During weeks 1-1 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.574, 2.156, and 2.059（P〈0.05） and the t values of the WIR, WOR, and MER at the edge of the lesions were 1.869, 2.058, and 2.057（P〈0.05）. During weeks 2-3 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.461, 2.098, and 2.739（P〈0.05） and the t values of the WIR, WOR, and MER at the edge of the lesions were 2.951, 2.625, and 2.154（P〈0.05）. During weeks 3-4 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.584, 2.107, and 2.869（P〈0.05） and the t values of the WIR, WOR, and MER at the edge of the lesions were 2.057, 2.637, and 2.951（P〈0.05）. During weeks 4-5 after treatment, the t values of the WIR, WOR, and MER at the center of the lesions were 2.894, 2.827, and 3.285（P〈0.05） and the t values of the WIR, WOR, andMER at the edge of the lesions were 3.45, 3.246, and 3.614（P〈0.05）. After the radiotherapy（500 c Gy）, the tumors shrank on the T1 WI, WIR, WOR, and MER; meanwhile, the PWI parameter gradually decreased and reached its minimum value.Conclusions： MR-DWI and MR-PWI can accurately and directly reflect the inactivation of tumor cells and the tumor hemodynamics in rabbit models with implanted pulmonary VX-2 carcinoma, and thus provide theoretical evidences for judging the clinical effectiveness of radiotherapy for the squamous cell carcinoma of the lung.

High glucose causes apoptosis of rabbit corneal epithelial cells involving activation of PERK-eIF2α-CHOP-caspase-12 signaling pathway

AIM: To investigate the effect of high concentration of glucose(HCG) on double stranded RNA-activated protein kinase-like ER kinase(PERK)-eukaryotic initiation factor-2α(eIF2α)-transcription factor C/EBP homologous protein(CHOP)-cysteine aspartate specific proteinase(caspase-12) signaling pathway activation and apoptosis in rabbit corneal epithelial cells(RCECs). METHODS: RCECs were treated by different concentrations of glucose for 0-48 h. The expressions of PERK, p-PERK, eIF2α, p-eIF2α, 78 k Da glucose-regulated protein 78(GRP78), CHOP, B-cell lymphoma 2(Bcl-2), B-cell lymphoma-2-associated X protein(Bax) and caspase-12 were determined by Western blot. Apoptosis was detected by TUNEL assay. Meanwhile, the function of PERK-eI F2α-CHOP-caspase-12 signaling pathway activation in high glucose-induced apoptosis was evaluated using PERK inhibitor, GSK2606414. RESULTS: HCG significantly promoted the expression of p-PERK, p-eIF2α, GRP78, CHOP, Bax and cleaved caspase-12 in RCECs(P<0.05), while remarkably decreased the expression of Bcl-2 and caspase-12(P<0.05), and the alterations caused by glucose were in concentration-and time-dependent manners. Meanwhile, PERK and eIF2α expressions were not affected in all groups(P>0.05). TUNEL assay showed that the apoptosis rate of RCECs in the HCG group increased significantly in contrast with that in the normal concentration of glucose or osmotic pressure control group(P<0.05), and the apoptosis rate increased with the increase of glucose concentration within limits(P<0.05). GSK2606414 down-regulated the expression of p-PERK and p-eI F2α in the HCG group(P<0.05), while still did not affect the expression of PERK and eIF2α among groups(P>0.05). Correspondingly, GSK2606414 also significantly reduced the apoptosis rate induced by high glucose(P<0.05). CONCLUSION: HCG activates PERK-eIF2α-CHOPcaspase-12 signaling pathway and promotes apoptosis of RCECs.

Characteristics of liver on magnetic resonance diffusion-weighted imaging:Dynamic and image pathological investigation in rabbit liver VX-2 tumor model

AIM: To investigate dynamical and image pathological characteristics of the liver on magnetic resonance (MR) diffusion-weighted imaging (DWI) in the rabbit VX-2 tumor model. METHODS: Forty New Zealand rabbits were included in the study and VX-2 tumor piece was implanted intrahepatically. Fifteen animals received two intrahepatic implantations while 25 had one intrahepatical implantation. DWI, T1- and T2-weighted of magnetic resonance imaging (MRI) were carried out on the 7th and the 14th d after implantation and DWI was conducted, respectively on the 21th d. Ten VX-2 tumor samples were studied pathologically. RESULTS: The rate of lump detected by DWI, T1WI and T2WI was 78.7%, 10.7% and 53.5% (χ2 = 32.61,P < 0.001) on the 7th d after implantation and 95.8%, 54.3% and 82.9% (χ2 = 21.50, P < 0.001) on the 14th d. The signal of most VX-2 tumors on DWI was uniform and it was equal on the map of apparent diffusion coefficient (ADC). The signal of VX tumors did not decrease on the 7th d after implantation, most of them slowly growing during the week following implantation without significant cell dying within the tumor. VX-2 tumors grew increasingly within 14 d after implantation but the signal of most VX-2 tumors on DWI or on the map of ADC was uniform or uneven and ADC of VX tumors decreased obscurely or slightly because tumor necrosis was still not obvious. On the 21th d after implantation, the signal of most VX-2 tumors on DWI or on the map of ADC was uneven because tumor necrosis was evident and ADC of VX-2 tumor necrotic areas decreased. The areas of viable cells in VX-2 tumors manifested a high signal on DWI and a low signal on the map of ADC. The areas of dead cells or necrosis in VX-2 tumors manifested low signals on DWI and low, equal or high signals on the map of ADC but they manifested high signals on DWI and on the map of ADC at the same time when the areas of necrotic tumor became liquefi ed or cystic. The border of tumors on DWI appeared gradually distinct and internal signals of tumor became progressively uneven. CONCLUSION: The manifestations of viable, necrotic and liquefi ed or cystic areas in VX-2 tumors on DWI are typical and DWI is of significant and potential values in clinical application in both the early detection and diagnosis of liver tumors.

Comparison of two different laparotomy methods for modeling rabbit VX2 hepatocarcinoma

AIM:To compare two different laparotomy methods for modeling rabbit VX2 hepatocarcinoma.METHODS:Thirty New Zealand rabbits were randomly divided into two groups:A and B.Group A was assigned a traditional laparotomy method(embedding tumor fragments directly into the liver with tweezers).Group B was subjected to an improved laparotomy method(injection of tumor fragments into the liver through a 15 G syringe needle).The operation time, incision length, incision infection rate, and mortality rate were compared between the two groups after laparotomy.Magnetic resonance imaging(MRI) was performed to evaluate tumor formation rates and the characteristics of the tumors 2 wk after laparotomy.RESULTS:The mean operation times for the two groups(Group A vs Group B) were 23.2 ± 3.4 min vs 17.5 ± 2.9 min(P < 0.05); the incision length was 3.3 ± 0.5 cm vs 2.4 ± 0.6 cm(P < 0.05); and the mortality rate after 2 wk was 26.7% vs 0%(P < 0.05); all of these outcomes were significantly different between the two groups.The incision infection rates in the two groups were 6.7% vs 0%(P > 0.05), whichwere not significantly different.MRI performed after 2weeks showed that the tumor formation rates in the two groups were 90.9%vs 93.3%(P>0.05).These rates were not significantly different between the two groups.The celiac implantation rate and abdominal wall metastasis rate in the two groups were 36.4%vs 13.3%(P<0.05)and 27.2%vs 6.7%(P<0.05),respectively,which were significantly different between the two groups.CONCLUSION:The tumor formation rates were not significantly different between the two methods for modeling rabbit VX2 hepatocarcinoma.However,the improved method is recommended because it has certain advantages.

Sphere-forming-like cells(squamospheres) with cancer stem-like cell traits from VX2 rabbit buccal squamous cell carcinoma

Previous studies have demonstrated that spheroid type cells grown under suspension culture conditions have cancer stem cell（CSC） traits in a number of cancers, but this phenomenon has not yet been reported in the VX2 rabbit oral cancer model. Hence, this study aimed to study the spheroid cells from VX2 rabbit buccal squamous cell carcinomas（SCCs） and assess their CSC characteristics. Five adult male New Zealand white outbred rabbits were used to generate VX2 rabbit buccal SCC. Sphere-forming cell culture was performed for the VX2 rabbit buccal SCC specimens. The self-renewal capability; cluster of designation（CD） 44, CD133, acetaldehyde dehydrogenase 1（ALDH1）, B cell-specific Moloney murine leukemia virus integration site 1（Bmi-1）, Nestin, octamer-binding transcription factor 4（Oct4）and reduced expression protein-1（Rex-1） expression with reverse transcription-polymerase chain reaction（RT-PCR）; chemoresistance to cisplatin and 5-fluorouracil; and in vivo tumorigenicity of spheroid cell transplantation in nude mice were evaluated to determine the CSC characteristics of the resulting spheroid cells. We successfully obtained spheroid cells from the VX2 rabbit OSCC tissues. The spheroid cells exhibited CSC traits, including the expression of CSC and stem cell markers（CD44, Bmi-1, Nestin, Oct4 and Rex-1）, capacity to generate new spheroid colonies within 1 week of reseeding from single-dissociated spheroid cells, chemoresistance capacity and generation of tumour xenografts（with histological features resembling those of the original VX2 rabbit buccal SCC） from the transplantation of 103 undifferentiated spheroid cells into nude mice. In summary, we demonstrated that spheroid cells with CSC cell traits can be derived from VX2 rabbit buccal SCCs, indicating that this animal cancer model is applicable for studying CSCs in human oral cancers.

Gene expression and MR diffusion-weighted imaging after chemoembolization in rabbit liver VX-2 tumor model

AIM: To investigate the dynamic characteristics and the correlation between PCNA, Bax, nm23, E-cadherin expression and apparent diffusion coefficient (ADC) on MR diffusion-weighted imaging (DWI) after chemoembolization in rabbit liver VX-2 tumor model. METHODS: Forty New Zealand rabbit liver VX-2 tumor models were included in the study. DWI was carried out periodically after chemoembolization. All VX-2 tumor samples in each group were examined by histopathology and Strept Avidin-Biotin Complex (SABC) immunohistochemical staining. RESULTS: The PCNA expression index in VX-2 tumors was higher than in the normal parenchyma around the tumor (P < 0.001). Nm23, Bax or E-caderin expression index in VX-2 tumors were lower than in the normal parenchyma around the tumor (all P < 0.001). PCNAand nm23 expression in the VX-2 tumor periphery first increased and then decreased (P < 0.001 and P = 0.03, respectively), while the expression of Bax and E-cadherin before and after chemoembolization was insignificant. When b-value was 100 s/mm2, there was a linear correlation between PCNA expression and ADC in the area of VX-2 tumor periphery (P < 0.001), and PCNA expression in VX-2 tumor periphery influenced the ADC. CONCLUSION: The potential of VX-2 tumor infiltrating and metastasizing decreases, while its ability to proliferate increases for a short time after chemoembolization. To some degree, the ADC value indirectly reflects the proliferation of VX-2 tumor cells.

MR diffusion-weighted imaging of rabbit liver VX-2 tumor

AIM: To investigate the implanting method of rabbit liver VX-2 tumor and its MR diffusion-weighted imaging (DWI) characteristics.METHODS: Thirty-five New Zealand rabbits were included in the study. VX-2 tumor was implanted subcutaneously in 14 rabbits and intrahepatically in 6 for pre-experiments. VX-2 tumor was implanted intrahepatically in 12 rabbits for experiment and three were used as the control group. DWI, T1- and T2-weighted of MR1 were performed periodically in 15 rabbits for experiment before and after implantation. The distinction of VX-2 tumors on DWI was assessed by their apparent diffusion coefficient (ADC) values. The statistical significance was calculated byanalysis of variance (ANOVA) of the randomized block design using SPSS10.0 software. RESULTS: The successful rate of subcutaneous implantation of VX-2 tumor was 29% (4/14) while that of intrahepatic implantation of it was 33% (2/6) in the preexperiment. The successful rate of intrahepatic implantation of VX-2 tumor in the experiment was 83% (10/12) and 15 tumors grew in 10 successfully implanted rabbits. The DWT signal of VX-2 tumor was high and became lower when the b value increased step by step. The signal of VX-2 tumor on the map of ADC was low. When the b value was 100 or 300 s/mm2, the ADC value of normal group and VX-2 tumor group was respectively 2.57±0.26, 1.73±0.31, 1.87±0.25 and 1.57±0.23 mm2/s. Their distinction was significant (F= 43.26, P＜0.01), the tumor ADC value between b values 100 and 300 s/mm2 wassignificant (Tukey HSP, P＜0.05) and the ADC value between VX-2 tumor and normal liver was also significant (Tukey HSP, P＜0.01). VX-2 tumor developed quickly and metastasized early to all body, especially to the lung, liver, lymph nodes of mediastinum, etc.CONCLUSION: The DWI signal of rabbit VX-2 tumor has its characteristics on MR DWI and DWI plays an important role in diagnosing and discovering VX-2 tumor.

Apoptosis of rabbit retinal cell after eyeball rupture

Objective:To establish rabbit eyeball rupture model by air gun in order to observe and analyze the early injury condition and reasons of retinal cell after eyeball rupture.Methods:Forty eight healthy rabbits were randomly divided into control group and 1,3,6,12 and 24 h after injury groups.After anesthesia,the rabbit eyeball rupture model was established by air gun. Then the early pathological changes of rabbit retina were observed,and apoptotic index(Al), oncosis index(OI),the relationship between the expression amounts of apoptosis-related genes and Al were analyzed.Results:Obvious pathological lesion appeared in retina 6 h after injury. Irreversible damage occurred 12-24 h after injury.The results of AI and OI indicated that the OI peak appeared 6 h after injury and then gradually declined,while the AI increased with the prolongation of time,and the AI was higher than OI in 12 h after injury.Immunohistochemical results indicated that there was no obvious bcl-2 protein expression change.Compared with the control group and the 3,6,12 and 24 h after the injury groups,the expressions of p53 and Caspase-3 were significandy improved and peaked at 12 h(P【0.01).Positive correlation existed among p53,Caspase-3 expression amount and cell apoptosis amount.Conclusions:Apoptosis and oncosis of visual cells are the main reasons of retinal cell injury.p53 and Caspase-3 are the important factors in promoting the retinal cell apoptosis after eyeball rupture.

Effects of WH-1 and WH-2 eye drops on experimental allergic uveitis in rabbits

Effects of WH-1 and WH-2 eye drops on experimental allergic uveitis(FAU)in rabbits induced by bovine serum albumin were clinically and pathologically observed in comparisonwith local drops of dexamethasone used as positive control.The results showed that WH-2 obvi-ously inhibited the inflammatory reactions while WH-1 did not.Protein concenwation in the aqueoushumor of WH-2-treated group of rabbits was 9.24±2.34mg/ml,which was notably lower(P【0.01)than both that of the untreated(14.33±3.14 mg/ml)and of the WH-1-treated(12.91±3.95mg/ml),but signifificantly higher than that of the dexame dexamethasone-treated goup(4.43±1.43mg/ml,P【0.01).The alleviation of the intraocular inflammatory reaction by therapy of WH-2eye drops was confimed pathologically.This study indicates that WH-2 has a positiveantiphlogistic effect on EAU in rabbits.

The Observation of the Influence of Vasectomy on Some Immunological Functions in Rabbits

To study the functional changes of immunological survcillsnce after vasectomy,we hzve dynamically obscrved the influence of vasectonly on the functions of T lymphocytes and macrophages of peripheral blood from vasectomized animals as the exPerimental model. The results showed that: (1) PHA and Con-A induced mitogenesis of T lymphocytes from the rabbits vasectomized for eight months was significantly inhibited (P<0.01) and recovered in rabbits vasectoomized for 12, 18 and 22 months respectively, (2) The IL -2 secretion from peripheral blood mononuclcar cells (PBMCs) in the rabbits vasectomized for 8 months was reduced remarkably (P<0.01) and reached the normal level in the rabbits vasectomlied for 12 months; (3) A significant correlation was found betwecn Con-A induced mitogenesis of T lymphocytes and the IL-2 secrcted from PBMCs (r=0.661, P<0.01); (4) The IL- 1 secrction form periPhoal blood macrophsges induced by LPS significantly increased in the rabbits vasectonlized for S months (P<0.02) and returned to the normal level in the rabbits vasectomized for 12, 18 and 22 months respectively, (5) The levl of plasma lysozyme increased significantly in the rabbits vasectomized for 8 months (P<0.05) and returned to the normal in the rabbits vasectomized for 12 months, (6) The level of IL- 1 secreted by the macrophages and the level of plasma lysozyme were closely. Correlated (r=0.576, P<0.001). The present results may provide an expeimantal evidence for the evaluating the functional changes of immunological surveillance in vasectomized animals.

Radiosensitivity of β-elemene on rabbit VX2 renal transplant carcinoma model

Objective To investigate the effects of low dosage of β-elemene on the radiosensitivity of rabbit VX2 renal transplant carcinoma model. Methods We took the rabbit VX2 renal transplant carcinoma as the model. Experimental rabbits were divided into three groups: the control group, the radiation group, and the radiation +β-elemene (radiosensitivity) group. The change of tumor was observed by Spiral CT and B ultrasound to compare its regrowth period. The tumor was measured by light microscopy and electron microscopy. Results The tumor in radiosensitivity group was restrained obviously and the sensitization enhancement ratio (SER) of β-elemene was 1.89. Different apoptosis was observed under transmission electron microscopy. Conclusion Low dosage β-elemene can enhance the radiosensitivity of rabbit VX2 renal transplant carcinoma model and induce the apoptosis of tumor cells, but the mechanism needs further study. It promotes apoptosis in mechanisms in vitro.