ObjectiveTo investigate the effect of cytoskeleton modification on the adipogenic differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor γ (PPARγ), 1ipoprotein lipase (LPL), and fatty acid binding protein (aP2). ResultsThe final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P<0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary group (P<0.05). ConclusionInhibition of F-actin polymerization can increase adipogenic differentiation of rat Achilles-derived TSCs in vitro, and cytoskeleton modification is a pre-requisite for TSCs differentiation into adipocytes, which might have important implications for the mechanism research of tendinopathy.
目的：了解大鼠脑出血后血肿周围组织细胞凋亡与神经元特异性烯醇化酶（NSE）的表达及大鼠神经功能缺损程度的关系。方法：用胶原酶注入到大鼠尾状核的方法制作脑出血模型。将大鼠分为脑出血、假手术组、正常组3组。采用苏木素伊红(HE) 染色、NSE免疫组织化学染色及TUNEL分别观察各组在脑出血后第6 h、12 h、24 h、48 h、72 h、5 d、7 d时血肿周围NSE及TUNEL的表达。用Longa评分法评价大鼠神经功能缺损程度。结果：大鼠在胶原酶注入6 h后形成稳定的血肿，在造模24～48 h神经功能缺损程度最重；6 h即见到TUNEL阳性细胞的表达，在48 h最明显；NSE从神经元中漏出弥散到细胞间隙也在48 h达高峰。结论：脑出血血肿周围凋亡与神经功能缺损及NSE的变化有关，凋亡可能在脑出血的神经损伤中起重要的作用。
摘要:目的:动态观察大鼠脑出血后血肿周围组织补体激活与细胞凋亡的规律。方法:用胶原酶注入到大鼠尾状核的方法制作脑出血模型。将大鼠分为脑出血、假手术组、正常组3组。采用苏木素伊红(HE) 染色、免疫组织化学染色及原位末端脱氧核苷酸转移酶介导的dUTP 缺口末端标记法(TUNEL)分别观察各组在脑出血后第6 h、12 h、24 h、48 h、72 h、5 d、7 d时血肿周围补体C3、促凋亡基因(Bax)、抑凋亡基因(Bclxl)及TUNEL的表达。结果补体C3的表达峰值在24~48 h;TUNEL、Bax蛋白表达术后12h增加,48~72 h达高峰,而Bclxl蛋白表达高峰在48h。结论:大鼠脑出血后血肿周围组织补体C3的表达增加与细胞凋亡的演变趋势一致,C3与凋亡有相关。Abstract: Objective: To study the complement activation and apoptosis regular genes changes in the tissues of the perihematoma of intracerebral hemorrhage (ICH) in rats. Methods: Intracerebral hemorrhage was induced in rats by injection of bacterial collagenase into the caudate nucleus. Histopathological changes were studied in 6 h,12 h, 24 h, 2 d, 3 d, 5 d, 7 d after the injection. The immunohistochemistry and TUNEL analysis were performed. The expression of complement factor C3, the TUNELpositive cells, the proapoptotic gene expression (Bax) and the antiapoptotic gene (Bclxl) were examined. Results: The expression of C3 increased to its maximum between 2448 h. The TUNELpositive cells and Bax protein expression increased gradually and reached the peak level between 4872 h. The Bclxl protein reached the peak level at 48 h. The correlation analysis showed that the quantity of C3 was positively related to that of the TUNELpositive cells, but the bax protein was not related to Bclxl protein. Conclusion: The expression of complement factor C3 may contributes to the nerve injury after cerebral hemorrhage and relate to the apotosis in the tissues surrounding the hametoma in rats.
摘要:目的:探讨卡配因抑制剂3（MDL28170）对新生大鼠缺氧缺血性脑损伤(HIBD)神经细胞凋亡的影响。方法:建立新生SD大鼠HIBD模型,治疗组于缺养缺血后即刻、2 h、4 h腹腔内注射MDL28170,对照组及手术组同时予生理盐水。缺氧缺血后24 h用免疫组化方法观察大脑皮质及海马CA1区Caspase3 蛋白表达、TUNEL法检测细胞凋亡,观察组织病理改变并计算海马神经元死亡数,透射电镜观察细胞超微结构。结果:缺氧缺血后24 h缺血侧大脑皮质及海马CA1区Caspase3和TUNEL阳性细胞数较对照组明显增加,透射电镜证实有凋亡细胞;MDL28170可减少阳性细胞数量,抑制神经元死亡,差异有显著性(Plt;0.05)。结论:MDL28170可通过抑制神经凋亡而对新生大鼠HIBD具有一定保护作用。Abstract: Objective: To investigate the effect of (Calpain inhibitor3) MDL28170 on neural apoptosis in a neonatal model of hypoxicischemic brain damage (HIBD). Methods: A neonatal model of HIBD was established, 7dayold SD rats were divided into three groups. The treatment group received MDL28170(ip) at 0 h,2 h,4 h after HI, whereas the other two groups were administered normal saline simultaneously. The expression of caspase3 (by immunohistochemistry), neural apoptosis (by TUNEL) in cortex and hippocampus ipsilateral to the insult were observed 24 h after HI; hippocampal CA1 neural loss and electromicroscopic changes were assessed at the same time. Results: Apoptotic body was observed by electromicroscopy. Caspase3 positive cells and apoptotic cells increased significantly in the ipsilateral cortex and hippocampal CA1 region compared to the control, and MDL28170 reduced the number of positive cells, attenuated CA1 neural loss with significance (Plt;0.05). Conclusion: It is suggested that MDL28170 may protect the brain of neonatal rats after HIBD by suppressing neural apoptosis.
Objective To explore the effect and mechanism of sleeve gastrectomy （SG） for type 2 diabetes mellitus （T2DM） in Goto-Kakizaki （GK） rats. Methods Thirteen male GK rats at 12 weeks of age were randomly divided into SG group （n=7） and sham operation group （SO group， n=6）， receiving SG surgery and sham operation respectively.Body weight， food intake in 24hours， fasting plasma glucose， plasma glucagon-like peptide-1 （GLP-1）， and plasma Ghrelin of rats in 2 groups were measured or tested before operation， 1， 4， 10， and 26 weeks after operation. In 10 weeks after operation， fecal energy content of rats in 2 groups was tested， in addition， oral glucose tolerance test （OGTT） and insulin tolerance test （ITT） were performed to investigate the glucose tolerance and insulin sensitivity. Results ①Body weight：there were no significant difference on body weight between the 2 groups （P＞0.05）. Compared with time point of before operation， the body weight of both 2 groups decreased in 1 week after operation （P＜0.01）， but increased in 10 weeks and 26 weeks （P＜0.01）. ②Food intake in 24 hours：compared with SO group， the food intake of SG group were lower in 4 weeks and 10 weeks after operation （P＜0.05）. Compared with time point of before operation， the food intake of SG group were lower in 1， 4， and 10 weeks after operation （P＜0.05）， but lower only in 1 week in SO group （P＜0.05）. ③Value of fasting glucose：compared with SO group， the value of fasting glucose in SG group were lower after operation （P＜0.01）. Compared with time point of before operation， the value of fasting glucose of SG group were lower after operation （P＜0.01）， but decreased in 1 week only in SO group （P＜0.01）. ④Level of serum GLP-1：compared with SO group， the levels of serum GLP-1 in SG group were higher in 4， 10， and 26 weeks after operation （P＜0.05）. Compared with time point of before operation， the levels of serum GLP-1 in SG group were higher in 4， 10， and 26 weeks after operation （P＜0.05）， but levels of serum GLP-1 in SO group didn’t change significantly （P＞0.05）. ⑤Level of serum Ghrelin：compared with SO group， the levels of serum Ghrelin in SG group were lower at alltime points after operation （P＜0.01）. Compared with time point of before operation， the levels of serum Ghrelin in SGgroup were lower at all time points after operation （P＜0.001）， but levels of serum Ghrelin in SO group didn’t change significantly （P＞0.05）. ⑥Areas under curves （AUC）：the AUC of OGTT and ITT test in SG group were both lower than those of SO group （P＜0.01）. Conclusion SG surgery can induce the level of fasting plasma glucose， and canimprove glucose tolerance and insulin sensitivity with significant changes of levels of plasma GLP-1 and Ghrelin， sugg-esting that SG surgery may be a potential strategy to treat patient with T2DM but without obesity or insulin resistance.
Objective To establish different kinds of reduced size liver transplantation model of rats， and to explorethe optimal marginal size of liver graft in orthotopic liver transplantation， in purpose of providing a kind of animal modelfor the study about mechanism and prevention measures of small-for-size syndrome. Methods One hundred and ninety-two rats were randomly divided into whole liver graft transplantation group （underwent whole liver graft transplantation），half liver graft transplantation group （the median lobe and right lobe of the liver were selected to be the graft）， small size liver graft transplantation group （the median lobe of the liver was selected to be the graft）， and extra-small size liver graft transplantation group （the median lobe and left lobe of the liver were reduced， and remained lobes were selected to be the graft）， each group enrolled 48 rats. After liver graft transplantation， 24 rats of each group were selected to observe the survival situation， 12 rats of each group were selected to measure portal venous pressure at time point of before operation，and 5， 15， 30， 45， and 60 minutes after transplantation. The other 12 rats of each group were test the level of alanine aminotransferase （ALT）. Results Seven-day survival rate of the whole liver graft transplantation group， half liver graft transplantation group， small size liver graft transplantation group， and extra-small size liver graft transplantation group was 100% （24/24）， 87.5% （21/24）， 37.5% （9/24）， and 0 respectively. Portal venous pressure of whole liver graft trans-plantation group was stable after opening the portal vein， although there was slight increase at prophase in half liver graft transplantation group， and then the portal venous pressure would let down， keeping stable at the later stage. But in small size liver graft transplantation group and extra-small size liver graft transplantation group， the portal venous pressure incr-eased and got the top at 15 minutes after opening the portal vein， and then induced， keeping stable during the 45-60 minutes.Portal venous pressure at the point of 5 （r=-0.942）， 15 （r=-0.947）， 30 （r=-0.900）， 45 （r=-0.825）， and 60 （r=-0.705）minutes after opening the portal vein were significantly related to liver graft size （P＜0.001）. The levels of ALT in wholeliver graft transplantation group and half liver graft transplantation group were both lower than those of small size livergraft transplantation group and extra-small size liver graft transplantation group （P＜0.05）， and levels of ALT in small size liver graft transplantation group was lower than extra-small size liver graft transplantation group too （P＜0.05）. Levelof ALT at 24 hours after transplantation were significantly related to liver graft size （r=-0.685， P＜0.001）. Conclusions The minimum graft volume/standard liver volume （GV/SLV） in reduced size liver transplantation in rat is 50%. The liver graft whose GV/SLV is 30%-35% should be considered as marginal size liver graft， and the liver graft whose GV/SLV less than 30% should be considered as extra-small size liver graft in the rat.
Objective To establish and evaluate the hind limb ischemia model in type 2 diabetic rats， and to providea platform for subsequent intervention experiment. Methods Fifteen SD rats were randomly divided into 3 groups：normal control group， diabetes group， and diabetic ischemia group， each group enrolled 5 rats. The 10 rats of later 2 groups were fed with high fat diet for 4 weeks， and then received intraperitoneal injection of streptozotocin （40 mg/kg） to establish type 2 diabetic model. Three days later， the rats of diabetic ischemia group underwent ligation of the bilateral common iliac artery to establish the hind limb ischemia model， but iliac artery of rats in other 2 groups didn’t received ligation. Ultrasound and color Doppler flow imaging detection was used to determine the peak velocity and acceleration time of femoral artery in rats of 3 groups in 2 weeks after operation， and triceps tissues and quadriceps tissues were collected to perform HE staining and SP staining for the observation on status of nutrition and vascular regeneration respectively. Results The peak velocity of femoral artery in rats of normal control group， diabetes group， and diabetic ischemia group were （22.49±3.02） cm/s， （17.36±2.60） cm/s， and （11.23±1.26） cm/s， the acceleration time were （0.080±0.009） s，（0.120±0.009）s， and（0.160±0.020） s， the arteriolar density measured by immunohistochemistry SP method were 6.80±0.84/HPF， 4.60±0.55/HPF， and 1.40±0.55/HPF respectively. The peak velocity of femoral artery and arteriolar density of rats in diabetic ischemia group were both lower （P＜0.05）， but acceleration time was longer （P＜0.05）. Results of HE staining showed that structure of triceps tissues was damaged， with infiltration of lots of inflamm-atory cells， which was worse than normal control group and diabetes group. Conclusion Method of high-fat diet in combination with small dose of streptozotocin can induce type 2 diabetic rat model， and hind limb ischemia model can be successfully established by ligation of common iliac artery on this model.
Objective To establish interstitial cells of Cajal （ICC） loss models caused by incomplete small intestinal obstruction in rats with modified method and verify it. Methods Modified method was used to establish the models， making the ring around the small intestine but not through it. Morphological changes were observed by general signs， pathological changes were tested by HE staining and transmission electron microscope （TEM）， and changes of ICC number were tested by immunohistochemistry staining. Results Success rate of this method was 56% （28/50）， weight loss happened compared with before operation in ileus group （P＜0.01）. Hyperemia and swelling were observed in ileus group， and gastric retention was obvious. Results of HE staining and TEM showed that there was obvious inflammatory change， and ICC reduced was observed by immunohistochemistry. Conclusion ICC loss models caused by incomplete small intestinal obstruction meet the basic performance， and can be used for further research.
Objective To establish a stable and reliable lung injury model caused by severe acute pancreatitis（SAP）in rats， which is helpful to study the acute lung injury （ALI）and acute respiratory distress syndrome （ARDS） induced by SAP.Methods Sixty Sprague-Dawley rats were randomized into ligature group （n=20）， traditional group （n=20），and sham operation group （n=20）. SAP model was established through retrograde injection of 5% taurocholic acid. After injection， the pancreatic duct of rats was ligated in ligature group， but not in traditional group. The lung damage and edema at 24 h after operaton and natural course of rats were observed.Results The ALI model of rats induced by SAP was established successfully in ligature group. The rats died of acute respiratory failure within 48 h in ligature group， the mortality was significantly higher than that in traditional group （100% vs.20%），P＜0.05. Pleural effusion occurred in four rats in ligature group， while no pleural effusion was found in rats in other two groups. The volume of ascites of rats in ligature group was （21.15±5.33） ml， which was more than that in traditional group 〔（7.75±2.66） ml〕，P＜0.05， while no ascites was found in rats in sham operation group. The level of serum amylase of rats in ligature group was （2 470.70±399.73） U/L，which was significantly higher than that in traditional group 〔（1 528.40±289.54） U/L〕 and sham operation group 〔（831.10±93.26） U/L〕，P＜0.001. The level of serum albumin of rats in ligature group was （6.90±1.66）g/L， which was significantly lower than that in traditional group 〔（13.10±0.99） g/L〕 and sham operation group 〔（16.20±0.92） g/L〕，P＜0.001.The lung wet-to-dry weight ratio （W/D） of rats in ligature group was 6.50±0.23， which was greater than that in traditional group （4.92±0.18） and sham operation group （4.61±0.16）， P＜0.001. The score of lung histopathologic of rats in ligature group was 29.25±1.07， which was significantly higher than that in traditional group （12.65±1.98） and sham operation group （0），P＜0.001. The score of pancreas histopathologic of rats in ligature group was 15.95±0.15，which was significantly higher than that in traditional group （13.75±0.66） and sham operation group （0.13±0.29），P＜0.001. Under transmission electron microscope， basement membrane of pulmonary capillary of rats in ligation group was destructive， the nuclei was dissolved， endothelial pinocytotic vesicles was functional active， and tight junctions between capillary endothelial cells were blurred and even ruptured. Moreover， tight junctions between alveolar epithelial cells were destructive. Pathological changes of lung ultrastructure of rats in ligation group were more severe than that in traditional group， while no pathological change of lung ultrastructure was observed in rats in sham operation group. Conclusions Injury process and pathogenesis of ALI or ARDS clinically caused by acute gallstone pancreatitis can be reproduced in this animal model， which is suitable to explore the related mechanisms of ALI caused by SAP and provides good animal model for the study of ALI caused by SAP.
Objective To establish chronic hindlimb ischemia model with suture-occluded method in rats， and then compare the effects of chronic hindlimb ischemia model with acute ischemia model. Methods Models of chronic hindlimb ischemia were established by using suture-occluded femoral artery method. The laser Doppler blood flow analysis and angiography were performed on day 7， 14， 28， 42， and 49 after operation， and then the rats were sacrificed after angiography， respectively， the quadriceps and gastrocnemius of contralateral and ipsilateral （surgical side） were gotten， which were tested by HE staining and α-actin immunohistochemistry staining， and then calculate arteriolar density. Results There were no lameness and limb necrosis after operation in chronic hindlimb ischemia models. Laser Doppler analysis found that chronic hindlimb ischemia models were still maintained in ischemia state on day 49 after operation compared with acute ischemic models. The resluts of HE staining showed no acute necrosis and muscle fibrosis in chronic hindlimb ischemia model group. Chronic hindlimb ischemia models after operation did not appear obvious lameness and limb necrosis. The arteriolar density of quadriceps femoris on day 7 after operation in chronic hindlimb ischemia models were less than that in acute hindlimb ischemia models （0.015 2 vs. 0.036 4）. Conclusions Compared with the commonly used acute ischemic models， the duration of arterial limb ischemia in chronic hindlimb ischemia rats， which were established by suture-occluded method， is longer and less likely to be affected by the compensatory mechanisms. So suture-occluded method can provide a new animal hindlimb ischemia model for further study of ischemia angiogenesis mechanism and treatment of severe lower extremity ischemia.