mSpag9肾脏特异性基因敲除小鼠模型的构建与鉴定

目的:为研究支架蛋白JLP在肾脏中的生理功能,建立小鼠精子相关抗原(mSpag9)肾脏特异性基因敲除小鼠模型,为研究该基因所发挥的重要生理功能提供材料和思路。方法:构建mSpag9基因重组载体,通过电转打靶转染胚胎干细胞(ES细胞),将筛选出的阳性ES细胞克隆,经显微注射至超排小鼠的囊胚腔后移植于受体小鼠,从子代筛选嵌合体小鼠,与携带flp位点C57/BL6N小鼠杂交后得到mSpag9flox/+基因敲除小鼠,自交筛选出mSpag9flox/flox小鼠,再与KSP-CreERT2小鼠杂交得到mSpag9flox/flox-Cre肾脏特异性基因敲除小鼠并进行鉴定,待小鼠成长至5周,使用Tamoxifen(10 mg/mL葵花籽油溶解)腹腔注射(0.04 mg/g)诱导Cre重组酶特异性表达于肾小管上皮细胞。结果:经PCR扫描后得到10株阳性ES细胞克隆,其中6株使用Southern Blot鉴定;经显微注射后得到4只雄性、5只雌性嵌合体小鼠,与flp小鼠杂交后得到9只mSpag9flox/+基因敲除小鼠,将自交得到的mSpag9flox/flox小鼠与KSP-CreERT2小鼠交配筛选出mSpag9flox/+-Cre+3只小鼠,将其与mSpag9flox/flox小鼠回交得到基因型为mSpag9flox/flox-Cre+小鼠,即为mSpag9肾脏特异性基因敲除小鼠。结论:该模型利用Cre-Loxp系统实现基因mSpag9的基因打靶,Tamxifen诱导基因mSpag9实现该基因在肾小管上皮细胞上敲除,为进一步研究mSpag9基因及支架蛋白在肾脏生理病理进展中所发挥的作用提供工具。     

巨噬细胞Lamtor2基因敲除小鼠对肺炎克雷伯菌易感性的研究

目的 通过建立Lamtor2巨噬细胞条件敲除小鼠感染肺炎克雷伯菌(Kp)模型,初步研究Lamtor2基因生物学功能及其在Kp感染过程中的相关机制.方法 将引进的Lamtor2flox/+小鼠自交,Lyz2-Cre小鼠与Lamtor2flox/+进行杂交,获得基因型为Lamtor2flox/floxLyz2-Cre-/-和Lamtor2flox/+Lyz2-Cre+/-的子代小鼠;将上述两种基因型子代小鼠进行杂交,得到对照组小鼠(Lamtor2flox/floxLyz2-Cre-/-)和Lamtor2条件性基因敲除小鼠(Lamtor2flox/floxLyz2-Cre+/-).提取小鼠脚趾基因组DNA,经聚合酶链式反应(PCR)扩增,通过琼脂糖凝胶电泳鉴定子代小鼠基因型并提取肺泡巨噬细胞,利用qRT-PCR和Western blot验证Lamtor2基因敲除效果.小鼠感染Kp,观察生存状况;体外采用RT-qPCR检测Kp感染肺泡巨噬细胞肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和Cxcl1表达水平,两组间比较采用t检验;Western blot验证iNOS的表达水平.结果 成功建立Lamtor2条件性基因敲除小鼠模型,子代存活且可育;与对照组小鼠比较,实验组小鼠在感染Kp后生存率降低;Lamtor2flox/floxLyz2-Cre+/-小鼠来源的肺泡巨噬细胞中的TNF-α(t = 17.54,P =0.003 2)、IL-1β(t = 15.37,P= 0.004 2)和 Cxcl1(t = 37.82,P = 0.000 7)表达水平降低,且iNOS表达下降.结论 构建了 Lamtor2条件性基因敲除小鼠,感染肺炎克雷伯菌后,小鼠的生存率下降;肺泡巨噬细胞iNOS表达降低.     

血液系统条件性DNMT 3B 基因敲除小鼠的构建及鉴定

目的 构建可在血液系统中条件性敲除DNMT 3B 基因小鼠并进行鉴定,为研究DNMT 3B 基因的生物学功能及其在血液肿瘤疾病的作用机制提供动物模型。方法 将引进的DNMT3Bflox/flox 小鼠与Mx1-Cre+ 小鼠进行杂交繁殖,得到基因型DNMT3Bflox/+Mx1-Cre+ 及DNMT3Bflox/+Mx1-Cre- 两种子代,再用这两种子代杂交产生子代小鼠,通过PCR 法鉴定子代小鼠的基因型;获得基因型为DNMT3Bflox/flox Mx1-Cre+ 的小鼠,通过腹腔注射pI-pC 共5 次诱导DNMT3B 敲除,然后用半定量PCR 方法鉴定DNMT 3B 基因敲除情况。结果 基因型鉴定确认在13 只子鼠中有2 只为DNMT3Bflox/flox Mx1-Cre+,经pI-pC 腹腔注射后,DNMT 3B 基因通过Cre/loxP 系统被成功敲除。结论 该方法成功构建可以在血液系统条件性敲除DNMT 3B 基因的小鼠,并进行敲除后鉴定。     

Treg中条件性Notch2基因敲除小鼠的构建及鉴定

目的 繁育和鉴定调节性T细胞(regulatory T cells,Treg)中条件性Notch2基因敲除小鼠,为Notch2在Treg中的功能提供研究工具;为Treg与免疫性疾病的发病关系及靶向治疗提供研究手段.方法 将引进的Notch2flox/flox小鼠和Foxp3-Cre+小鼠进行饲养和杂交;将繁育得到的第一代小鼠进行相互交配,再得到第二代小鼠.通过聚合酶链反应(polymerase chain reaction,PCR)鉴定出具有Notch2flox/flox Foxp3-Cre+基因型的小鼠,再通过该小鼠进行繁育.通过流式细胞术分选出野生型小鼠和Notch2flox/flox Foxp3-Cre+小鼠体内的Treg,Western blot检测小鼠Treg中Foxp3、Notch2、NICD2蛋白的表达,苏木精-伊红(hemotoxylin-eosin,HE)染色观察小鼠肺脏等器官的形态学变化.结果 该方法成功构建了在Treg中敲除Notch2基因的小鼠.Western blot结果显示Notch2flox/flox Foxp3-Cre+小鼠体内的Treg中Notch2、NICD2、Foxp3蛋白表达明显下降(P<0.05),HE染色显示该小鼠肺脏等器官出现明显炎症细胞浸润.结论 本研究成功构建了Treg中条件性敲除Notch2基因的实验小鼠,为Treg与免疫性疾病的发病和靶向干预研究奠定了基础.     

血管内皮细胞特异性敲除cdc42基因的杂合子小鼠与非基因敲除小鼠在急性肺损伤肺组织病理改变以及肺微血管通透性变化的比较

目的比较血管内皮细胞特异性敲除cdc42基因的杂合子小鼠与非基因敲除小鼠在急性肺损伤肺组织病理改变和肺微血管通透性变化的差异。方法 cdc42flox/flox小鼠与血管内皮细胞特异性表达cre重组酶的小鼠杂交,取其基因型为cdc42flox/+Cre+/-的子代小鼠与cdc42flox/flox小鼠回交,得到基因型分别为Cdc42flox/+Cre+/-、Cdc42flox/+Cre-/-、Cdc42flox/floxCre-/-的小鼠,其中Cdc42flox/+Cre+/-为血管内皮细胞特异性敲除cdc42基因的杂合子小鼠,其余两种同一窝出生的非基因敲除小鼠作为对照组,在各组小鼠气道内滴入LPS,制成急性肺损伤模型,比较各组小鼠在肺组织病理改变、病理评分、肺湿干重比值、肺微血管通透系数等指标变化的差异。结果血管内皮细胞特异性敲除cdc42基因的杂合子小鼠急性肺损伤模型在肺组织病理改变、病理评分、肺湿干重比值、肺微血管通透系数等方面与对照组小鼠比较无明显统计学差异。结论血管内皮细胞特异性敲除cdc42基因杂合子小鼠与非基因敲除小鼠比较,在急性肺损伤中肺组织病理改变和肺微血管通透性的变化无明显差异。     

胰岛 β 细胞中 PP2Acα 基因失活小鼠模型的建立与初步表型分析

摘要 目的：建立胰岛 β 细胞特异性失活 PPAcα 基因小鼠模型,对其表型进行初步观察。方法：通过 Ins2-Cre 与 PP2Acαflox/flox 小鼠交配,获得胰岛 ? 细胞特异性失活 PP2Acα 基因小鼠 (PP2Acαflox/fIox:Ins2-Cre)。PCR 和 Western Blot 鉴定 PP2Acα 基因第二外显子敲除小鼠 (KO)。4 月龄时行腹腔注射葡萄糖耐量试验 (IPGTT),以 PP2Acαflox/flox 小鼠为对照。结果：1,PP2Acα 转录本在 KO 小鼠短于对照小鼠。2,KO 小鼠胰岛中 PP2Ac 蛋白水平较对照小鼠显著下降。3,IPGTT 结果显示：4 月龄 KO 小鼠 30 min、60 min、120 min 时血糖明显高于对照小鼠 (P<0.05)。结论：成功建立胰岛 ? 细胞特异性失活 PPAcα 基因小鼠模型,4 月龄 PP2Acαflox/fIox:Ins2-Cre 小鼠糖耐量受损。     

成熟成骨细胞中敲除基因fgfr1小鼠的制备

目的 制备在成熟成骨细胞中敲除基因fgfr1的特定细胞阶段性基因敲除小鼠.方法 从美国NIH引进SPF级fgfr1条件性基因敲除(fgfr1flox/flox)小鼠,进行饲养、繁殖和PCR鉴定;fgfr1flox/flox小鼠与在成熟成骨细胞表达Cre重组酶的OC-Cre小鼠交配,获得在成熟成骨细胞中敲除一个基因拷贝的杂合子小鼠;杂合子小鼠之间交配,或者杂合子小鼠与fgfr1flox/flox小鼠交配,可以获得在成熟成骨细胞中敲除fgfr1基因的小鼠fgfr1△/△/OC-CreTG/ .结果 成功制备在成熟成骨细胞中敲除fgfr1的细胞特异性敲除小鼠.结论 成功引进fgfr1flox/flox小鼠,应用合理的基因条件性敲除繁殖策略,获得在成熟成骨细胞中敲除fgfr1的基因敲除小鼠.     

HIF-1α条件性基因敲除嵌合体小鼠的获得

目的应用Cre/loxP系统建立HIF-1α条件性基因敲除小鼠模型。方法电击法将构建好的HIF-1α条件性基因敲除载体转染入小鼠ES细胞,用G418与Ganciclovir对转染的ES细胞进行正负筛选,经Southern杂交鉴定得到阳性ES细胞克隆后,将已发生正确重组的ES细胞显微注射入小鼠囊胚腔,对子代鼠进行基因型鉴定。结果经G418与Gan-ciclovir筛选培养得到490个阳性克隆,用5′端探针进行Southern杂交鉴定获得1株发生同源重组的ES细胞克隆,经显微注射,得到5只由基因敲除ES细胞和供体囊胚细胞共同发育而来的嵌合体小鼠。结论成功得到基于Cre/loxP系统的HIF-1α条件性基因敲除嵌合体小鼠模型。     

Tex101条件性基因敲除小鼠模型的建立和表型鉴定

目的建立Tex101（Testis expressed gene101）条件性基因敲除小鼠模型并进行表型鉴定。方法构建针对小鼠Tex101基因2、3号外显子的重组载体,通过电穿孔的方法转染胚胎干细胞（ES细胞）,用正负筛选法筛选阳性ES细胞并进行PCR鉴定。利用显微注射把发生正确同源重组的ES细胞注射进C57BL／6J小鼠囊胚,移人受体小鼠子宫。将得到的嵌合体小鼠与C57BL／6J雌鼠交配获得Tex101-LoxP转基因小鼠。该小鼠与EⅡa—Cre转基因小鼠杂交,通过子代自交获得Tex101条件性基因敲除（cKO）小鼠并进行初步分析。结果得到2只Tex101cKO雄鼠。冰冻切片观察发现Texl01cKO雄鼠睾丸形态和大小与野生型小鼠相比无明显差异。结论成功建立了Tex101条件性基因敲除小鼠模型,并初步验证了该基因对雄性小鼠睾丸大小和形态无明显影响,为进一步研究Tex101基因功能奠定了基础。     

神经系统SARM1条件性敲除小鼠的构建及其应用

目的：构建SARM1基因在神经细胞中条件性敲除小鼠模型,为探究SARM1基因在神经系统中的功能提供研究工具。方法：运用ES细胞打靶的方式构建SARM1flox/flox转基因小鼠,并将其与表达Nestin-Cre重组酶的工具鼠进行杂交以产生神经元特异性SARM1基因敲除小鼠。然后使用PCR对敲除小鼠进行基因型鉴定,使用Western blot验证基因敲除效果。通过对小鼠进行旷场与高架十字迷宫试验来评估小鼠的情绪行为和焦虑水平。结果：SARM1蛋白主要表达于中枢神经系统,在神经元中高表达。PCR结果表明,成功构建了神经元特异性SARM1基因敲除小鼠。Western blot结果显示,与正常小鼠对比,SARM1Nestin-CKO小鼠的脑组织中SARM1蛋白表达显著降低（P＜0.05）,并且SARM1基因敲除不影响小鼠的正常生长发育,也不影响脑组织的一般形态和神经细胞的数量。同时发现该条件性敲除小鼠不存在明显的焦虑样表型。结论：成功构建了SARM1基因在神经细胞中特异性敲除的小鼠动物模型,可为探讨SARM1基因在神经精神疾病中的作用提供重要研究平台。     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

Development on Adjustable Calibration Marker for Shock Wave Focus

Shock wave lithotripsy （SWL） is a treatment of choice for upper urinary stones. However, this procedure is inappropriate for obese patients because the focus is often unable to reach the target owing to the limited focal distance in shock wave source. Although treating such patients in a blast path may increase the application length of shock wave source, it＇s difficult to find this path on the lithotripter monitor. For this reason, we invented an adjustable calibration marker in order to set an effective focus in the shock wave hath.     

Study on the TCM Pathogenesis of Angiopathic Complications of Type II Diabetes

Excess production of reactive oxygen species(ROS)of mitochondrion mediated by hyperglycemia is the common pathogenesis of angiopathic complications of diabetes.TCM holds that the damp from the dysfunction of spleen.kidney and liver is the causative factor of complications of diabetes.This is similar to the mechanism of Ros resulting in angiopathic complications of diabetes.When the angiopathic complications of type II diabetes mellitus(T2DM)are difierentiated as caused by turbid damp in TCM can be explained as ROS.Since the obstruction of pathogenic damp in channels and collaterals is said to be the main pathogenesis,the treating principle should be dissolving the damp to remove the obstruction.     

Leptin, Ghrelin and TNF-α before and after Hypo-caloric Traditional Chinese Diet and Auricular Acupuncture

INTRODUCTION Obesity is a complex emergent problem, which can be possibly solved not only by the diet but also by the life style and promotion of a constant physical exercise. 1, 2 No doubt careful attentions must be given to the nutritional condition of obese people, the dietary habits, the somatic build (i.e. distribution of fat mass) and the organic functions linked to formation of the fat mass. All the parameters should be constantly monitored before, during and after a diet treatment. 3, 4, 5     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

Relationship between Dysglycemia and Carotid Atherosclerosis in Tibetan Population

People with dysglycemia are at high risk for atherosclerotic diseases. This study aims at investigating the atherosclerotic vascular damage in dysglycemia and its metabolic origin in Tibetan population.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.     

Luo Lingjie's Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.