IL-18基因导入对小鼠胶原诱导关节炎作用的研究

目的观察IL-18基因导入对小鼠胶原诱导性关节炎（CIA）的治疗作用。方法在CIA小鼠发病早期,将IL-18表达质粒pCAGGS-IL-18（pIL-18）经肌肉注射导入CIA小鼠体内,对照组给予空白质粒pCAGGS,3周后同量加强治疗1次。用关节炎评分评估CIA小鼠的病情,用组织学染色评价膝关节的软骨和骨破坏,用RT-PCR和real-time PCR法测定CIA小鼠髌骨及邻近滑膜组织的细胞因子及转录因子表达水平。结果与对照组相比,治疗组能有效抑制胶原诱导性关节炎小鼠的关节炎评分,阻止关节软骨破坏,虽然髌骨及邻近的滑膜组织IL-18的mRNA表达没有明显改变,但TNF-α及IL-17A的mRNA表达明显降低,IL-10的mRNA表达明显增加,同时,Th2型转录因子GATA-3的表达明显增高,Th2型转录因子T-bet及Th17型转录因子ROR-γt的表达无明显改变。结论与IL-18的细胞因子治疗不同,单独IL-18表达质粒的基因导入即可对CIA小鼠达到治疗作用。该作用通过提高GATA-3的表达,增加Th2型细胞因子IL-10,降低Th1型细胞因子TNF-α及Th17型细胞因子IL-17A的表达来实现。     

FIZZ1与动脉粥样硬化关系探讨

目的 探讨FIZZ1在ApoE基因敲除鼠粥样斑块表达情况并探讨其可能的表达细胞.方法 C57BL/6J ApoE基因敲除鼠及C57BL/6J野生型小鼠各9只,分别喂食高脂饲料及普通饲料,24周后处死小鼠,自主动脉根部至腹主动脉离断整个血管,石蜡包埋后作连续切片,行HE染色及FIZZ1免疫组化.检测血管斑块内FIZZ1表达情况,体外模拟粥样斑块内Th2型细胞因子环境,检测斑块内FIZZ1表达是否与Th2型细胞因子刺激平滑肌细胞或巨噬细胞有关.结果 免疫组化可见FIZZ1在粥样硬化斑块内明显表达,Th2型细胞因子可以刺激巨噬细胞FIZZ1表达,但不能刺激平滑肌细胞FIZZ1表达.结论 正常血管壁内未见FIZZ1表达,ApoE基因敲除鼠粥样斑块表达FIZZ1,动脉粥样硬化斑块内Th2型细胞因子刺激巨噬细胞可能为FIZZ1表达机理之一.     

TGF-β1对皮肤组织特异性Smad4基因敲除小鼠组织培养影响的研究

目的应用组织块培养的方法，观察TGF-β1及其受体抑制剂SB-431542对皮肤组织特异性Smad4基因敲除小鼠与野生型小鼠表皮细胞增殖的影响。方法皮肤组织特异性Smad4基因敲除小鼠与野生型小鼠，制成背部烫伤模型。伤后5d取创面边缘皮肤组织，剪成1mm×2mm皮块，行组织块培养。实验分为6组，分别是：（1）Smad4基因敲除组（A组）；（2）Smad4基因敲除＋SB431542组（B组）；（3）杂合子组（C组）；（4）杂合子＋SB431542组（D组）；（5）野生型组（E组）；（6）野生型＋SB431542组（F组）。B、D、F组加入5×10^-4 MSB431542，作用1h。各组均加入5ng／ml TGF-β1，于37℃，5％C02条件下培养。于培养的第0、8、16、24、32、40、48小时，加入10μM BrdU标记液，标记1h。10％中性甲醛固定。病理切片，免疫组化染色，计算BrdU阳性率。结果TGF-β1可以降低杂合子和野生型小鼠表皮细胞PCNA阳性率和BrdU掺入率，TGF-β1作用于野生型小鼠16h，表皮细胞BrdU掺入率即有明显降低，但对基因敲除组小鼠无该作用，在皮肤组织块培养条件下，预先应用受体抑制剂SB431542作用1h，则TGF-β1的这种作用不再存在。结论TGF-β1对表皮细胞生长具有抑制作用，其受体拮抗剂SB-431542能拮抗这种抑制作用。     

脑卒中病理变化的机制：PSD93在血小板活化因子性神经元损害中的作用

目的：探讨PSD93基因是否参与血小板活化因子所致的神经元毒性损伤，以研究血小板活化因子(platelet activating factor，PAF)神经性损伤的分子学机制。方法：实验于2003-05／2004-05在南京鼓楼医院神经科研究室和美国约翰霍普金斯大学Dr．Tao实验室进行。用原代神经元细胞培养系统，PAF处理wild type(野生型)和PSD93 Knockout(基因敲除)小鼠皮质神经元；propidium iodide／calcein AM染色进行细胞凋亡测定。荧光显微镜下数码相机拍照。结果：0．10，0．30和0．60μmol／LPAF对野生型和PSD93基因敲除型两种皮质神经元细胞具有明显的神经元毒性(P&;lt;0．05)。基因敲除型型的小鼠皮质神经元细胞凋亡明显低于野生型(P&;lt;0．01)。结论：PSD93基因敲除型可抑制PAF所致的神经元损害。     

β-内啡肽对胶原诱导关节炎大鼠的免疫调节作用

目的研究β-内啡肽（β-END）对胶原诱导性关节炎（CIA）大鼠的免疫调节作用。为探索β-END治疗类风湿关节炎（RA）提供实验依据。方法采用尾根部皮内多点注射天然Ⅱ型胶原（CⅡ）的方法免疫雌性Wistar大鼠（60只）。建立CIA模型。随机取CIA成模大鼠（5只／组）于初次免疫后第14-35天。给予不同浓度的β-END腹腔内注射，定期进行临床、实验室、影像学及病理指标评估。结果不同剂量β-END（0．1、1、5nmol隔日1次共2周）治疗后CIA大鼠临床、实验室、影像学及病理指标明显缓解；正常鼠β-ENO给药5nmol×2周后重要脏器功能、组织学未见明显异常。结论生理浓度的β-END体内可缓解CIA鼠关节局部及全身免疫炎性反应。这使β-END成为有潜力的治疗RA的制剂。     

12-脂氧化酶基因敲除对1型糖尿病肾病小鼠肾小球内纤维连接蛋白表达的影响

目的探讨12-脂氧化酶（12-lipoxygenase,12-LO）对1型糖尿病肾病小鼠肾小球内纤维连接蛋白（Fibronectin,FN）表达的影响。方法用链脲佐菌素（Streptozotocin,STZ）诱导1型糖尿病模型。野生型和12-LO基因敲除C57BL/6小鼠随机分成4组：野生型对照组;12-LO基因敲除组;野生型糖尿病组;12-LO基因敲除糖尿病组。采用RT-PCR和免疫组化方法分别检测肾小球内FN mRNA和蛋白表达的变化。结果与野生型对照组比较,野生型1型糖尿病小鼠血糖（P〈0.01）、肾重/体重比值（P〈0.01）和24小时尿白蛋白明显增高（P〈0.01）,但12-LO基因敲除糖尿病小鼠尿白蛋白（P〈0.05）、肾重/体重比值（P〈0.05）明显低于野生型糖尿病小鼠。基础情况下,野生型和12-LO基因敲除小鼠肾小球内FN表达无差异。与野生型对照组相比,野生型糖尿病小鼠肾小球内FN表达明显增加（P〈0.01）,但12-LO基因敲除可阻止糖尿病小鼠肾小球FN表达的增加（P〈0.05）。结论 12-LO基因敲除延缓1型糖尿病肾病小鼠蛋白尿的进展,降低肾小球内FN表达。     

血管活性肠肽对胶原诱导关节炎大鼠治疗的作用初探

目的探讨血管活性肠肽(VIP)对胶原诱导性关节炎(CIA)大鼠的免疫调节作用,为应用VIP治疗类风湿关节炎(RA)提供理论依据。方法采用尾根部皮内多点注射法注射天然Ⅱ型胶原(CⅡ),免疫雌性Wistar大鼠(60只),建立CIA模型。随机取CIA成模大鼠(5只/组)于初次免疫后14~35 d,腹腔内注射不同浓度的VIP。定期进行临床、实验室、影像学及病理指标评估。结果不同剂量VIP(0.1、1、5 nmol隔日1次,共2周)治疗后CIA大鼠,其临床、实验室、影像学及病理指标明显缓解;正常鼠于VIP给药5 nmol 2周后重要脏器功能、组织学未见明显异常。结论生理浓度的VIP体内可缓解CIA鼠关节局部及全身免疫炎性反应,5 nmol 2周以内的体内治疗量基本安全,提示VIP可作为治疗RA有潜力的制剂。     

腺苷A1受体敲除小鼠戊四氮点燃后脑组织病理形态学变化

目的:通过观察腺苷A1受体敲除小鼠戊四氮点燃后脑组织病理形态学变化,探讨腺苷A1受体的神经保护作用。方法:腺苷A1受体基因敲除纯合型(-/-)小鼠20只纳入敲除鼠组,腺苷A1受体基因敲除野生型小鼠20只纳入野生型组,C57BL/6小鼠10只纳入对照组。敲除鼠组和野生型组小鼠制作戊四氮点燃癫痫模型,于点燃成功后24 h、30 d采用尼氏染色观察各组小鼠海马及皮质神经元的形态结构变化。结果:野生型组小鼠点燃后皮质和海马神经元损伤较敲除鼠组出现晚、范围小,损伤程度轻。结论:腺苷A1受体对癫痫发作小鼠具有神经保护作用。     

异基因骨髓间充质干细胞移植对小鼠胶原性关节炎CD4＋CD25＋调节性T细胞表达的影响

目的：探讨异基因骨髓间充质干细胞（MSCs）移植对小鼠胶原性关节炎 CD4＋、CD25＋调节性 T 细胞表达的影响。方法选取40只 C57BL/6（H-2b）小鼠,随机分为正常对照组、Ⅱ型胶原性关节炎（CIA）模型组、MSCs 移植治疗组、甲氨蝶呤治疗阳性对照组,每组10只小鼠。除正常对照组外,其余各组弗氏完全佐剂＋Ⅱ型胶原诱导小鼠建立 CIA 小鼠模型。分离骨髓单个核细胞后,进行 MSCs 细胞分选及鉴定。美国 BD 公司 FAcscal-iburTM 型流式细胞分析仪对小鼠骨髓 MSCs 进行分选和鉴定。 MSCs 移植采用尾静脉注射,所含细胞数为2×106。移植后第42天全部处死动物观察不同分组小鼠的关节症状及肿胀度,采用流式细胞术检测 CD4＋ CD25＋浓度,收集数据并做统计分析。结果 MSCs 移植治疗组小鼠关节的肿胀度,与正常对照组比较,差异无统计学意义（P＞0．05）。 MSCs 移植组 CD4＋ CD25＋调节性 T 细胞的表达,与甲氨蝶呤治疗阳性对照组比较,差异有统计学意义（P＜0．05）;与正常对照组比较,差异无统计学意义（P＞0．05）。结论 MSCs 移植治疗小鼠 CIA ,能显著改善关节的肿胀度,并明显上调 CD4＋ CD25＋表达,故 MSCs 可作为一种新的替代细胞来源用于类风湿性关节炎的移植治疗。     

β-内啡肽对胶原诱导关节炎大鼠的免疫调节作用

目的研究β-内啡肽(β-END)对胶原诱导性关节炎(CIA)大鼠的免疫调节作用,为探索β-END治疗类风湿关节炎(RA)提供实验依据。方法采用尾根部皮内多点注射天然Ⅱ型胶原(CⅡ)的方法免疫雌性Wistar大鼠(60只),建立CIA模型。随机取CIA成模大鼠(5只/组)于初次免疫后第14~35天,给予不同浓度的β-END腹腔内注射,定期进行临床、实验室、影像学及病理指标评估。结果不同剂量β-END(0.1、1、5 nmol隔日1次共2周)治疗后CIA大鼠临床、实验室、影像学及病理指标明显缓解;正常鼠β-END给药5 nmol×2周后重要脏器功能、组织学未见明显异常。结论生理浓度的β-END体内可缓解CIA鼠关节局部及全身免疫炎性反应,这使β-END成为有潜力的治疗RA的制剂。     

Minnie mice

In determining the size of an animal or plant, it has long beenthought that size itself is in some way measured and monitored.Experimental manipulation of rates of cell proliferation orcell size during growth results in organs and/or organisms ofthe normal size that may consist of fewer larger cells, or morenumerous smaller cells.¹ As Day and Lawrence² have pointed out,when looking for answers to questions about how this is achieved,the intuitive response is to search for mechanisms that countcell divisions or add up cell numbers. Ploidy is clearly     

Adiponectin knockout mice

Here we investigated the biological functions of adiponectin, a fat-derived hormone, by disrupting the gene encodes it in mice. Adiponectin knockout mice (KO) exhibited severe diet-induced insulin resistance with reduced IRS-1-associated P13-kinase activity in muscle. KO also revealed severe neo-intimal thickening in response to vascular-injury and hypertension induced by salt diet. Carbon-tetrachloride induced severe liver fibrosis in KO with the elevated gene expression of growth factors. These phenotypes in KO were reversed by viral-mediated production of adiponectin. Our results indicate that adiponectin should be one of key molecule of the metabolic syndrome and may be a new therapeutic target for the metabolic syndrome.     

Immunomodulation in offspring mice after neonatal immunostimulation of mothers or newborn mice

Mothers of offspring Balb/c mice were stimulated after birth by two substances, a bacterial lysate (LAB) and a chemical, diethyldithiocarbamate (DETC). Anti-sheep red blood cell (SRBC) antibodies were studied after immunization of stimulated mothers or offspring. An increase of anti-SRBC was observed in LAB-stimulated mothers, but these antibodies were decreased in their offspring before weaning. Sometimes, these antibodies were increased in LAB-stimulated newborn mice. DETC stimulation of mothers induced an elevation of antibody response in mothers and newborns. The same results were obtained in previous investigations where the pregnant mother was stimulated with the same agents.     

GIP receptor knockout mice

Gastric inhibitory polypeptide(GIP) is a gastrointestinal peptide hormone, which is secreted from duodenal endocrine K cells after absorption of glucose or fat. It is well known as an incretin. To determine the further role of GIP in vivo, we generated GIP receptor-knockout mice. The mice showed higher blood glucose levels with impaired initial insulin response after oral glucose load. Even after high-fat diet, knockout mice lack compensatory insulin secretion, and showed no hyper-insulinemia. Moreover, knockout mice fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Therefore, GIP directly links glucose tolerance and over-nutrition to obesity and it is a potential target for the treatment for the metabolic syndrome.     

Gelatinase activities in wounds of healing-impaired mice versus wounds of non-healing-impaired mice

The gelatinases, matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9), have been implicated in different aspects of wound repair. However, little is known about MMP-2 and MMP-9 activity in animal models of impaired wound healing. We sought to compare serial gelatinase activities for 25 days after full-thickness excisional wounds in genetically diabetic healing-impaired mice and their nondiabetic non-healing-impaired littermates. Wound samples were frozen, homogenized, clarified by centrifugation, and analyzed on zymography gels, and MMP bands were quantitated relative to a conditioned media standard from HT-1080 cells. Gelatinase activity in both diabetic mice and nondiabetic mice increased after the mice were wounded. However, levels of latent gelatinases peaked earlier in the diabetic wounds, and there was more active MMP-2 and MMP-9 in the wounds of the diabetic mice than in the wounds of the nondiabetic mice. Because the higher gelatinase activity in the wounds of the diabetic mice was similar to the higher levels of gelatinase reported in difficult-to-heal wounds such as ulcers and burns, this diabetic mouse model may be useful for studies of these proteinases and their inhibitors in impaired wound healing.