周质的

RESULTSdiabetic rats showed typical symptoms involving elevated serum glucose and weight loss (2)The rats in DM+VaD group reacted slower and made more error numbers in Y-maze than the rats in VaD group did, with a prolonged total reacting time in a whole day.(3)More neurons with serious damages, such as edema, ischemia or pyknosis could be found in DM+VaD group than in VaD group. More disarrangement of cone neurons, obvious glia proliferation, and more neuron apoptosis were found in CA area of hippocampus. CONCLUSIONS (1)2-VO in STZ-induced diabetic rats succeeded in establishing VaD animal models.(2) The cognitive dysfunction induced by VaD rats had been deteriorated by diabetes mellitus.(3)Morphology evidences proved that diabetes mellitus aggravated brain damages induced by VaD.PART II Effect of diabetes mellitus on the cholinergic nervous systemin CA1 area of hippocampus of VaDOBJECTIVE To estimate the role of cholinergic nervous system in hippocampus during the process of cognitive dysfunction aggravated by diabetes mellitus in DM+VaD group. METHODS Observe the changes of ChAT protein by immunohistochemistry stain. Chose three rats randomly from each group at each pointat two week, four week, and eight week, then sepa

经腹腔注射链脲佐菌素诱导慢性实验性糖尿病，1周后永久性结扎双侧颈总动脉(2-VO)制作VaD模型记录术后2周、4周和8周各组大鼠的体重及血糖；应用Y-迷宫检测空间定向学第二军医大学博士论文中…丈摘…要1ia；一色恤~﹁染澎糕行HE染色观察组织病理学孪叱；胶质细胞原纤维酸性蛋白protellZ，Cf冰尸标记观察星形胶质细舱的激活情况、橄声软公点娜育票票众糯黑橄居端粗默孺篇筑价井袱第二部分糖尿病对血管性痴呆大鼠海马c心区胆碱能神经系统的影响探讨海马CAI区胆碱能神经系统在糖尿病加重确D一大鼠认知障碍中所实验动物及分组同第一部分，应用免疫组织化学染色方法现察海马CAI{介狱牛第晕军l袭拔常博粗{论义

Histological observation: Four weeks after implantation, porcine cancellous bone scaffold/bone marrow stromal cells complex appeared new bone formation and scaffold's degradation, which significantly increased in 8 weeks; In 12 weeks, new bone continued to increase and changed gradually to mature bone, a small amount of the Havers system was seen, the trabecular bone was not typical, capillary proliferated obviously, few residual scaffolds were seen, and no inflammatory cells were observed.

组织学观察：猪松质骨支架/骨髓基质干细胞复合体植入后4周开始有新骨生成，支架开始降解；8周时新骨的形成及支架的降解都明显增加；12周时新骨继续增加，逐渐向成熟骨组织转变，可见少量哈佛系统，但骨小梁尚不典型，毛细血管增生明显，少量支架残留，未见炎症细胞。

The unoperated sides of the treated animals also served as controls. Six normal rats were treated as normal control group. Three different siRNA plasmid solution containing RC2-Ⅰ, MAFbx-Ⅱ, CON (50μl , 0.8μg/μl)was injected and transfected by electroporation as methods mentioned above, respectively. The changes of RC2 and MAFbx mRNA levels and RC2 protein levels after 3 days were determined by real-time quantitative PCR and Western blot, respectively. On postoperative 2, 3 and 4 weeks, the rate of wet muscle weight preservation, mean diameter of muscle fiber and mean cross-section area of muscle fiber and muscle protein content were checked and then compared between group CON and group RC2 or group MAFbx, respectively. The differences between groups were analyzed by one-way ANOVA. Ultrastructural changes of muscle fiber were observed at 2, 3, 4 weeks postoperation.Results GFP plasmid was efficiently deliverd into muscle by electroporation and robust GFP expression in muscle could be observed more than three weeks. Histology shows that injected plasmid DNA diffuses extensively in muscle tissue.

1、健康雌性SD大鼠18只，随机分为电穿孔组和非电穿孔组，每组9只，制作右下肢趾长伸肌失神经支配模型；EP组为将质粒pEGFP-N1溶液50μl(0.8μg／μl)注射入右趾长伸肌后，立即于两侧腱腹交接处给予电穿孔，电穿孔参数为：电场强度为200V／Cm，脉冲100μs，频率1Hz，施加10次脉冲；NEP组仅质粒pEGFP-N1溶液注射；转染后1、2、3周，荧光显微镜下观察趾长伸肌中GFP的表达情况，转染后1周行Western印迹检测趾长伸肌中GFP蛋白的表达情况，检测和优化体内转染效率。2、健康雌性SD大鼠78只，随机分为失神经对照组、RC2基因治疗组(RC2组)，MAFbx基因治疗组，每组24只，制作右下肢趾长伸肌失神经支配模型，余6只为正常组；分别将含CON、RC2、MAFbx基因的siRNA重组质粒注射入趾长伸肌，之后给予电穿孔，方法同上；治疗后3天实时定量PCR和Western印迹检测各组中RC2或MAFbx基因的mRNA和蛋白的表达变化，治疗后2、3、4周检测各组肌湿重维持率、肌细胞直径和肌细胞截面积，肌细胞超微结构变化以及肌纤维中蛋白含量变化。

When initial NO3--N was leached, the correlation coefficients between soil initial NO3--N and mineral N extracted by CaCl2 before aerobic incubation with raygrass nitrogen uptake were decreased to 0.613 and 0.607, respectively, and both reached 5% significantly level. However, the correlation coefficients between mineralizable N extracted by aerobic incubation, soil initial mineral N and mineralizable N extracted by aerobic incubation, N0 and soil initial mineral N and N0 with raygrass nitrogen uptake were raised obviously, and all reached 5% and 1% significant level.

以不包括土壤起始NO3--N盆栽试验植物吸氮量为参比，通气培养前CaCl2淋洗起始NO3--N和起始矿质氮与五期黑麦草地上部氮素累积量间的相关性尽管有所降低，但相关性仍达5%显著水平，相关系数分别为0.613和0.607；而通气培养30周矿化氮素、土壤起始矿质氮+通气培养30周矿化氮素、N0及N0+起始矿质氮与五期黑麦草地上部吸氮量的相关系数却明显提高，相关系数分别为0.718，0.782，0.688 和0.640，均达5%或1%显著水平。

Ovarian stromal flow PSV, EDV and perifollicular vascularity are highly interrelated with ovarian response. Ovarian response will be improved when ovarian blood velocity increases and perifollicual blood flow is abundant. Follicular fluid VEGF level was obviously higher in poor responder group, which suggests VEGF be secreted increasing for compensating due to reduced ovarian stromal blood velocity and fewer perifollicular vascularity.

卵巢基质内血流的PSV、EDV和卵泡周血流分布、卵泡液VEGF水平与卵巢的反应性密切相关，卵巢基质内血流速度升高、卵泡周边血流丰富，有助于提高卵巢的反应性；低反应卵巢组卵泡液VEGF水平显著升高，推测是由于其卵巢基质内血流速度低，卵泡周血管分布少，导致VEGF代偿性分泌增加。

The results are unsure for experimental use;The rabbit's corneas that were removed with upper-half of corneal limbal epithelium lamella and erased the center corneal epitheliums were transparent with intact corneal epithelium;In the approach,the corneal and limbal epitheliums were burned with a cotton swab socked in 1 mol/L NaOH,there were 4 rabbits' corneal stroma happened perforation or ulcer and symblepharon,and the other one presented corneal epithelium phenotype.This is an applicable method to create the pathological model of corneal limbal stem cell total deficiency.

结果表明，处理后4周，全周角膜缘上皮板层手术切除，中央角膜上皮层用1 mol/L NaOH擦除的5只试验家兔角膜表面全部血管化、结膜化，未发生睑球粘连，角膜基质胶原纤维完整未见溃疡、穿孔等病变，细胞印迹学检查为结膜表型，可作为实验性角膜缘干细胞移植的病理模型；全周角膜缘上皮板层手术切除，中央角膜上皮用生理盐水擦除的5只试验家兔，有2只为结膜表型，另3只为角膜表型，观察期内结果不稳定；半周角膜缘上皮板层手术切除，中央角膜上皮层用生理盐水擦除的5只试验家兔，角膜表面透明，全部为角膜表型；直接用1 mol/L NaOH擦除角膜缘和中央角膜上皮的试验家兔，有4只角膜基质胶原纤维断裂、溶解，并伴有严重的溃疡、穿孔、睑球粘连等病变，不能用于移植试验，另1只角膜表面透明，未见结膜和新生血管长入，细胞印迹学检查为角膜表型。

Fig 1 At the experimental side, two weeks after operation (HE ×100) Many pieces of cartilage cells and spindle-shaped mesenchymal cells were found, by light microscope Fig 2 At the experimental side, twelve weeks after operation (HE ×100) Medullary cavity had formed, which contained hemoblasts and adipocytes, but the trabculae was not typical, by light microscope Fig 3 At the experimental side, twenty-four weeks after operation (HE ×100) The typical cancellous bone had formed, by light microscope Fig 4 At the control side, twenty-four weeks after operation (HE ×100) A large part of CXB was degraded, resorbed and replaced by fibrous tissue, but there was'not any new formed bone, by light mocroscope

图1 实验侧术后2周(HE ×100)镜下可见很多软骨细胞条索和团块生成及大量梭形间充质细胞图2 实验侧术后12周(HE ×100)镜下可见有髓腔形成，内含有造血和脂肪细胞，骨小梁尚不典型图3 实验侧术后24周(HE ×100)镜下可见形成典型的松质骨结构图4 对照侧术后24周(HE ×100)镜下可见大部分CXB被降解吸收，为纤维组织替代，无新骨形成

By use of site mutation strategy and PCR technology, we obtained the gene P12X3C that includes full length P1, 2A, 3C and a part of 2B and 3B and the gene P12X3C3D that includes full length P1, 2A, 3C, 3D and a part of 2B and 3B. After being digested by restriction enzyme respectively, the gene P12X3C and the gene P12X3C3D were cloned into the pcDNA3. 1 and pTARGET expression vector that were digested by the same enzyme. Recombinant plasmids were checked by restriction enzyme analysis and nucleic acid sequencing. Further more, recombinant plasmids were transfected into BHK-21 cells by using lipoid. The proteins of foot-and-mouth disease virus , which were expressed in BHK-21 cells, were confirmed by sandwich-ELISA and fluoroscopy, and the capsid of FMDV was tested by electron microscope. In order to evaluate enhanced immune response of guinea pigs against FMDV, DNA vaccines which were designed to produce viral capsids lacking infectious viral nucleic acid and contained the gene P12X3C and the gene P12X3C3D were injected respectively with FMDV 3D protein which was expressed in Pichia Pastoris Secreted expression System and purified or with pcDNA3. 1/IFN which includes the gene IFN-α of cattle. Subsequently, Recombinant plasmids were injected to cattles with or without pcDNA3. 1/IFN. Anti-FMDV antibodies were detected by ELISA, and the T lymphocyte proliferation response was tested by MTT assay, neutralization antibodies titers were analyzed by micro-neutralization assay.

为研制带有O型口蹄疫病毒(Foot-and-Mouth Disease Virus，FMDV)China99株结构蛋白基因及多个非结构蛋白基因的DNA疫苗，本研究通过定点突变方法和PCR扩增方法，获得包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C以及部分2B、3B编码基因的片段P12X3C和包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C、3D以及部分2B、3B编码基因的片段P12X3C3D，将获得的基因片段直接/酶切后与同样处理的真核表达质粒连接，分别得到重组质粒pcDNA3.1/P12X3C和pcDNA3.1/P12X3C3D、pTARGET/P12X3C3D；对重组质粒进行序列测定、分析，并将重组质粒分别转染BHK-21细胞，通过双抗体夹心ELISA方法和间接免疫荧光标记方法检测细胞中FMDV抗原的表达，用电子显微镜观察病毒空衣壳的组装；为评价重组质粒作为DNA疫苗对实验动物及本动物的免疫效果，将重组质粒经肌肉注射方法接种豚鼠，并与酵母表达的纯化FMDV China99株3D蛋白及带有牛α干扰素的真核表达质粒pcDNA3.1/IFN分别/同时免疫，第二次免疫后第三周豚鼠攻以1OOID〓或1000ID〓的O型FMDV China99株；随后将质粒pcDNA3.1/P12X3C、pcDNA3.1/P12X3C3D与带有牛α干扰素的真核表达质粒pcDNA3.1/IFN同时免疫牛，三周后经牛舌皮攻以10〓ID〓的O型FMDV China99株。

By use of site mutation strategy and PCR technology, we obtained the gene P12X3C that includes full length PI, 2A, 3C and a part of 2B and 3B and the gene P12X3C3D that includes full length PI, 2A, 3C, 3D and a part of 2B and 3B. After being digested by restriction enzyme respectively, the gene P12X3C and the gene P12X3C3D were cloned into the pcDNA3.1 and pTARGET expression vector that were digested by the same enzyme. Recombinant plasmids were checked by restriction enzyme analysis and nucleic acid sequencing. Further more, recombinant plasmids were transfected into BHK-21 cells by using lipoid. The proteins of foot-and-mouth disease virus, which were expressed in BHK-21 cells, were confirmed by sandwich-ELlSA and fluoroscopy, and the capsid of FMDV was tested by electron microscope. In order to evaluate enhanced immune response of guinea pigs against FMDV, DNA vaccines which were designed to produce viral capsids lacking infectious viral nucleic acid and contained the gene P12X3C and the gene P 12X3C3D were injected respectively with FMDV 3D protein which was expressed in Pichia Pastoris Secreted expression System and purified or with pcDNA3.1/lFN which includes the gene IFN-a of cattle. Subsequently, Recombinant plasmids were injected to catties with or without pcDNA3.1/IFN. Anti-FMDV antibodies were detected by ELISA, and the T lymphocyte proliferation response was tested by MTT assay, neutralization antibodies liters were analyzed by micro-neutralization assay.

为研制带有O型口蹄疫病毒(Foot-and-Mouth Disease Virus，FMDV)China99株结构蛋白基因及多个非结构蛋白基因的DNA疫曲，本研究通过定点突变方法和PCR扩增方法，获得包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C以及部分2B、3B编码基因的片段P12X3C和包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C、3D以及部分2B、3B编码基因的片段P12X3C3D，将获得的基因片段直接／酶切后与同样处理的真核表达质粒连接，分别得到重组质粒pcDNA3.1／P12X3C和pcDNA3.1／P12X3C3D、pTARGET／P12X3C3D；对重组质粒进行序列测定、分析，并将重组质粒分别转染BHK-21细胞，通过双抗体夹心ELISA方法和间接免疫荧光标记方法检测细胞中FMDV抗原的表达，用电子显微镜观察病毒空衣壳的组装；为评价重组质粒作为DNA疫苗对实验动物及本动物的免疫效果，将重组质粒经肌肉注射方法接种豚鼠，并与酵母表达的纯化FMDV China99株3D蛋白及带有牛α干扰素的真核表达质粒pcDNA3.1／IFN分别／同时免疫，第二次免疫后第三周豚鼠攻以100ID_(50)或1000ID_(50)的O型FMDV China99株：随后将质粒pcDNA3.1／P12X3C、pcDNA3.1／P12X3C3D与带有牛α干扰素的真核表达质粒pcDNA3.1／IFN同时免疫牛，三周后经牛舌皮攻以10~4ID_(50)的O型FMDV China99株。

Results 1、 Generally, we can see the original blue and white, shiny, no cracks in the articular surface of the cartilage after the stress increases gradually yellow, surface roughness, cracks appear; when the pressure decreases, the yellowing, rough, the color of the fracture restore gradually and become shiny.2、the shiny smooth surface can be seen under a light microscope, formation, cell distribution, tidy, clear the level of cartilage at the articular surface stress increases, the surface roughness changes, defects, disordered cells, uneven dyeing ; when the articular surface of the pressure gradually decreased, the cartilage gradually repair and the surface of cells at the surface appear only disorder.3、immunohistochemical observation can be seen throughout the observation period, cartilage cells are type Ⅱ collagen expression and expression after 3 weeks gradually weakening, when the seventh week begin to strong gradually.4、 electron microscopy shows that when stress increases the articular surface, the cartilage cells became flat, the cytoplasm in the endoplasmic reticulum, Golgi apparatus decreased with collagen disorders; and when stress decreases the articular surface, cartilage cells gradually returned normal, cytoplasm in the endoplasmic reticulum, Golgi body gradually restore quantity; collagen fibers with a gradual rules.

结果：①大体观察可见到原本蓝白色、有光泽、无裂纹的软骨在关节面压力增大后，逐渐呈灰黄色，表面粗糙，出现裂隙；当压力逐渐减小后，变黄、粗糙、有裂隙的软骨颜色逐渐恢复，变得有光泽②光镜下可见表面光滑、平整，细胞分布均匀、整齐，层次清楚的软骨在关节面压力增大后，表面变粗糙、缺损，细胞排列紊乱、染色不均；当关节面压力逐渐减小后，软骨表面逐渐修复，细胞仅在表层排列紊乱③免疫组织化学观察可见整个观察期内软骨细胞胞浆内均有Ⅱ型胶原表达，术后3周内表达逐渐变弱，从第7周时开始逐渐变强。④电镜下可见当关节面压力增大后，软骨细胞逐渐变扁，胞质中内质网膜、高尔基体减少，胶原排列紊乱；当关节面压力减小，软骨细胞形态逐渐恢复正常，胞质中内质网膜、高尔基体数量逐渐恢复；胶原纤维排列逐渐有规则。

I can not make it blossom and suits me

我不能让树为我开花

When temperatures are above approximately 80 °C discolouration of the raceways or rolling elements is a frequent feature.

当温度高于 80 °C 左右时，滚道或滚动元件褪色是很常见的特征。