流式细胞仪检测细胞凋亡的虚拟仿真实验

针对"流式细胞仪检测细胞凋亡实验"的实验成本高且实验内容复杂等问题,本文提出构建高度仿真的虚拟实验教学系统来弥补教学中的不足。根据流式细胞仪检测细胞凋亡实验的实验特点和操作流程,阐述了虚拟实验教学系统的建设内容和开发思路。使用3D Max建模软件和Unity3D开发引擎,搭建一个具有沉浸感和交互感的虚拟仿真实验系统。同时开发配套的web端实验辅助教学平台,学生可以进行在线学习、在线测评和互动等操作。本着虚实结合的原则,旨在帮助学生初步了解流式细胞仪,熟悉流式细胞仪检测细胞凋亡实验的基本原理和操作流程,提高教学效果。

健脾养胃方对人胃腺癌SGC7901细胞株的生长抑制和诱导凋亡作用的实验研究

目的:通过观察健脾养胃方对人胃腺癌SGC7901细胞株的生长抑制和诱导凋亡作用,探讨该方对人胃腺癌SGC7901细胞株生物学行为的影响。方法:通过四噻唑氮蓝(MTT)比色分析法,观察健脾养胃方对SGC7901细胞的增殖抑制作用;采用transwell小室法,研究健脾养胃方对SGC7901细胞侵袭力的影响;以Annexin-V/PI双染色,通过流式细胞仪,检测健脾养胃方对人胃腺癌SGC7901细胞凋亡的影响及其对细胞周期的影响。结果:健脾养胃方能抑制人胃腺癌SGC7901细胞增殖,以高剂量作用最为明显;健脾养胃方作用细胞24h后,肿瘤细胞侵袭力降低,高剂量组肿瘤细胞侵袭的细胞数最少;健脾养胃方作用细胞48h后可以诱导细胞凋亡;健脾养胃方可诱导SGC7901细胞G2/M期周期阻滞。结论:健脾养胃方对人胃腺癌SGC7901细胞株的生长抑制和诱导凋亡作用,是该方抑制胃癌细胞的重要机制。

体外震波对骨微血管内皮细胞生理功能的影响

背景:体外震波作为一种特殊类型的外源性物理作用信号,通过刺激、激活内皮细胞上机械力学信号感受器,激活细胞内特殊信号传导系统,调控内皮细胞基因表达,对内皮细胞生理功能产生影响。目的:观察不同能流密度及作用次数体外震波对股骨头骨微血管内皮细胞成血管能力、迁移能力及抗凋亡作用的影响。方法:从关节置换患者股骨头获取股骨头骨松质,分离、培养、传代培养股骨头骨微血管内皮细胞。通过血管性血友病因子、CD31血管内皮细胞标记物抗体进行免疫荧光鉴定。根据体外震波不同能流密度(低0.03 m J/mm2,高0.11 mJ/mm2)及不同作用次数(400次,800次)对细胞进行分组。通过细胞3D培养实验观察内皮细胞成血管能力,划痕实验观察内皮迁移能力,流式细胞仪检测内皮细胞凋亡情况。结果与结论:(1)从股骨头松质骨中分离、培养出的细胞均能表达血管性血友病因子、CD31,表明这些细胞具有血管内皮细胞的特征,阳性率接近100%;(2)体外震波作用于股骨头骨微血管内皮细胞提高其体外成血管能力及迁移能力,低能流密度组促进成血管能力和细胞迁移能力显著优于高能流密度组,高能流密度组内随着作用次数的增加促成血管能力下降;(3)对于激素诱导股骨头骨微血管内皮细胞凋亡有保护作用;(4)结果说明,体外震波对股骨头骨微血管内皮细胞生理功能的影响与作用能流密度及作用次数相关。

CCR5亲和短肽抗肿瘤活性研究

目的探讨趋化因子受体CCR5亲和短肽(AFDWTFVPSLIL)对肿瘤生成作用的影响。方法通过MTT实验、细胞凋亡实验检测短肽对血管内皮细胞生长的影响,通过鸡胚绒毛尿囊膜(CAM)实验检测短肽对血管生成的影响,通过体内抑瘤实验检测短肽对实体瘤生长的作用。结果短肽能通过诱导人脐静脉血管内皮细胞的凋亡来抑制内皮细胞的生长;并能抑制CAM膜的血管生成。短肽也能在体内抑制小鼠B16黑色素瘤的生长活性,其抑瘤率达68.6%。结论初步证明短肽具有抗肿瘤生成作用,且这种作用可能是通过抑制肿瘤的血管生成来实现。

Experimental Study on Double Blocking of T Lymphocytes Apoptosis Induced by Fas/FasL

Objective： To block the apoptosis of T lymphocytes induced by Fas/FasL in order to establish a method of the large-scale preparation of large amounts of tumor-specific cytoxic T-lymphocytes （CTL）. Methods： Liver cancer cells and tumor infiltrating lymphocytes （TIL） were isolated from FasL positive fresh specimens, and co-cultured. Specific CTL were activated and prepared in the presence of the co-stimulation of monoclonal antibody CD28. Then the blocking and activation of apoptosis of T lymphocytes was activated by soluble Fas receptor, which was detected by cytometry and DNA ladder test simultaneously. The apoptosis-blocking effect was compared with the control group. Furthermore, the changes of T cell proliferation and killing activity were detected by the method of ^3H thymidine incorporation and ^51Cr release test. Results： There was a significant increase in apoptosis rate in unblocking group compared with blocking group and quiescent group, with the unblocking group of 47.82%±0.13%, quiescent group of 3.76%±0.25%, and the blocking group of 8.22%±0.26% respectively （P〈0.01）. T cell-ladder appeared in unblocking group by DNA ladder test. Both the killing ability and proliferation rate of T cells were significantly increased after blocking. There was significant difference among blocking group, unblocking group and quiescent group （P〈0.01）. Conclusion： With this method we obtained large amounts of tumor-specific T lymphocytes, which was able to kill liver cancer cells effectively.

电针对大鼠肾缺血再灌注损伤的疗效及对肾组织中caspase-3表达的影响

目的:探讨电针治疗对肾缺血再灌注大鼠肾功能及组织中Caspase-3表达的影响。方法:将30只清洁级健康SD雄性大鼠随机分为假手术组、缺血再灌注组(IRI组)和电针组,每组10只。采用右肾摘除,左肾肾蒂夹闭缺血45min后再灌注的方法制备动物模型。电针组于造模成功后给予电针肾俞和涌泉穴治疗。各组均于术后24h采用全自动生化分析仪检测血清尿素氮(BUN)和肌酐(Scr),观察肾组织病理形态学改变,TUNEL法检测肾组织细胞凋亡率和免疫组化检测肾组织细胞中Caspase-3的表达。结果:肾缺血再灌注后大鼠血清BUN和Scr明显升高,肾组织切片显示有明显的病理改变,肾组织细胞凋亡率及Caspase-3阳性细胞表达显著升高。与缺血再灌注组比较,电针组大鼠BUN和Scr显著下降,肾组织细胞凋亡率显著降低,Caspase-3阳性细胞的IOD值显著降低。结论:电针对IRI所致肾功能损害具有显著的保护作用,能够抑制Caspase-3蛋白表达,具有抑制细胞凋亡的作用,该作用可能是电针对IRI后肾脏保护作用的重要机制之一。

Experimental Study on Apoptosis in Leukemia Cells Induced by Econazole

To investigate apoptosis in mouse leukemia cell (WEHI-3) induced by Econazoleand its mechanism. Methods: Apoptosis induced by Econazole was examined by flow cytometry. Freecalcium ([Ca^(2+])i) was determined by Fura-2 fluorescein load technique. The protein was isolatedfrom endoplasmic reticulum of WEHI-3 cells, and then the expression of caspase-12 and caspase-7 wasdetected by Western blot. Results: WEHI-3 cells exhibited typical change of apoptosis when they weretreated by Econazole. [Ca^(2+)]i was significantly higher in Econazole-treated group than incontrol group. The expression of caspase-12 and caspase-7 was increased with the increase ofEconazole concentration. Conclusion: Caspase-12 may play a key role in WEHI-3 apoptosis induced byEconazole.

How the Bcl-2 family of proteins interact to regulate apoptosis

Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.

Mitochondrial protection by low doses of insulin-like growth factor-Ⅰin experimental cirrhosis

AIM： To characterize the mitochondrial dysfunction in experimental cirrhosis and to study whether insulin-like growth factor-Ⅰ （IGF-Ⅰ ） therapy （4 wk） is able to induce beneficial effects on damaged mitochondria leading to cellular protection. METHODS： Wistar rats were divided into three groups： Control group, untreated cirrhotic rats and cirrhotic rats treated with IGF-Ⅰ treatment （2 μg/1O0 g bw/d）. Mitochondrial function was analyzed by flow cytometry in isolated hepatic mitochondria, caspase 3 activation was assessed by Western blot and apoptosis by TUNEL in the three expedmental groups. RESULTS： Untreated cirrhotic rats showed a mitochondrial dysfunction characterized by a significant reduction of mitochondrial membrane potential （in status 4 and 3）; an increase of intramitochondrial reactive oxigen species （ROS） generation and a significant reduction of ATPase activity. IGF-Ⅰ therapy normalized mitochondrial function by increasing the membrane potential and ATPase activity and reducing the intramitochondrial free radical production. Activity of the electron transport complexes Ⅰ and Ⅲ was increased in both cirrhotic groups. In addition, untreated cirrhotic rats showed an increase of caspase 3 activation and apoptosis. IGF- Ⅰ therapy reduced the expression of the active peptide of caspase 3 and resulted in reduced apoptosis. CONCLUSION： These results show that IGF- Ⅰ exerts a mitochondrial protection in experimental cirrhosis leading to reduced apoptosis and increased ATP production.

Dynamic expression of apoptosis-related genes during development of laboratory hepatocellular carcinoma and its relation to apoptosis

AIM： To explore the expression of p53, bcl-2, bax, survivin and the cell apoptosis during the development of tree shrew hepatocellular carcinoma （HCC）, the relationship between expression of these genes, its impact on HCC development, and its relation to cell apoptosis. METHODS： Tree shrew HCC was induced with aflatoxin B1 （AFB1）, and regular biopsy of liver tissues was carried out and the biopsy tissues were collected during cancer inducement. Liver biopsy tissue and HCC tissue were collected from 35 pre-cancerous experimental animals at wk 30 and 60 and at the 30^th, 60^th, and 90^th -wk. Liver biopsy tissues were collected from 13 blank control animals at wk 30, 60, and 90. Expression of p53, bcl-2, bax, and survivin at each stage was examined by immunohistochemistry method. Apoptotic cells were detected in situ by the terminal deoxynucleotidyl transferase-mediated nick end labeling （TUNEL） technique. RESULTS： The apoptosis rate of normal hepatic cells was extremely low, whereas it increased during the formation of HCC. Expression of the apoptosis-related genes p53, bcl-2, bax, and survivin during the formation of HCC presented an increasing tendency. Expression of p53 did not noticeably relate to that of bcl-2, bax, and survivin, whereas expression of bcl-2 and bax was closely related. In HCC, p53 did not present a distinct relation to cell apoptosis, whereas its high level expression was probably related to liver cell proliferation. Survivin negatively correlated apoptosis index, and its overexpression could inhibit cell apoptosis. CONCLUSION： Apoptosis-related genes p53, bcl-2, bax, and survivin are all related to the occurrence of HCC. The anti-apoptosis effect of bcl-2 is influenced by bax, and ratio bcl/bax reflects more correctly the extent of cell apoptosis.

Tumor necrosis factor-alpha induces apoptosis of enterocytes in mice with fulminant hepatic failure

AIM: To explore the alterations of intestinal mucosa morphology, and the effects of tumor necrosis factor a (TNFα) on enterocyte apoptosis in mice with fulminant hepatic failure (FHF). METHODS: Liver damage was induced by lipopolysaccharide (LPS)/TNF-α in D-galactosamine (GaIN) sensitized BALB/c mice. There were 40 mice in normal saline (NS)-treated group, 40 mice in LPS-treated group, 40 mice in GaIN-treated group, 120 mice in GaIN/ LPS-treated group and 120 mice in GaIN/ TNFα-treated group. Each group was divided into five subgroups of eight mice each. Serum samples and liver, intestinal tissues were respectively obtained at 2, 6,9,12 and 24 h after administration. Anti-TNFa monoclonal antibody was injected intravenously into GaIN/LPS-treated mice. Serum TNFα levels were determined by enzyme linked immunosorbent assays (ELISA). Serum ALT levels were determined using an automatic analyzer. The intestinal tissues were studied under light microscope and electron microscope at 2, 6, 9,12 and 24 h in mice with fulminant hepatic failure, respectively. Enterocyte apoptosis was determined by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) method. The expression of tumor necrosis factor receptor 1 (TNFR1) in intestinal tissue was tested by immunohistochemistry Envision Two Steps. RESULTS: Gut mucosa was morphologically normal at all time points in all groups, but typical apoptotic cells could be seen in all experimental groups under electron microscope. Apoptosis rate of gut mucosal epithelial cells were significantly increased at 6, 9 and 12 h, peaked at 12 h in mice with fulminant hepatic failure. TNFa induced apoptosis of enterocytes in mice with FHF. The integrated OD (IOD) levels of TNFa receptor 1 protein expressed in the intestine of mice with GaIN/LPS and GaIN/ TNFα induced FHF at 2, 6, 9, 12 and 24 h after GaIN/LPS and GaIN/TNFα administration were 169.54±52.62/905.79±111.84,11 350.67±2 133.26/28 160.37±4 601.67, 25 781.00±2 277.75/122 352.30±49 412.40, 5 241.53±3 007.24/ 49 157.93±9 804.88, 7 086.13±1 031.15/3 283.45±127.67, respectively, compared with those in control groups (with NS, LPS and GaIN administration, respectively). IOD level of TNFR1 changed significantly at 6, 9 and 12 h after GaIN/LPS and GalN/TNFa administration. The expression of TNFR1 protein was significantly higher at 9 h after GaIN/LPS and GaIN/TNFα administration than that in control groups. Protein expression of TNFR1 was positively correlated with enterocyte apoptosis. CONCLUSION: TNFα can induce apoptosis of enterocytes in mice with FHF. Anti-TNFα IgG can inhibit this role.

Action of induced hypertension and apoptosis: an experimental study in rat

Objective To study the retinal tissue degeneration of intraocular hypertension experimentally induced in acute and chronic way. Methods In the acute model, pressure elevation was quickly induced by a needle in the anterior chamber and the retinal reaction was studied at 1,2,4,5,7,10 days after treatment. The tissue damage with chronic hypertension,induced by the cauterization of two episcleral veins,was studied at 1 and 2 month after the treatment. The TUNEL method and Caspade 3a immunochemical study evidenced the apoptosis mechanism. The NADPH-diaphorase reaction identified the Nitric Oxide, ( NO) producing cells. Results In the acute model,the immunohistochemical study evidenced that the apoptosis was an early death mechanism for ganglionar cells. The activity of Nitric Oxide Synthase ( NOS) didn' t show a significant activation toward the retinal tissue of control. The chronic hypertension model indicated an increase in the NOS signal, meaning an activation of this enzyme in particular bear the vascular vessels, showing the neuroprotective and not only cytotoxic effect of the NO. The TUNEL and Caspase 3a studies indicated that the apoptosic mechanism started in different times, the immunohistochemical reaction showed its immediately beginning or its later activation caused by the chronic damage. Conclusions The opportunity to have a clear vision of the beginning and causing factors of cell degeneration in hypertension damage could permit the study on different substances that act on apoptosis , on NOS mechanism and on synaptic transmission inhibiting or deviating the retinal tissue degeneration and particularly the ganglionar cells death in glaucoma.

THE ROLES OF bcl-2 GENE FAMILY IN THE PULMONARY ARTERY REMODELING OF HYPOXIA PULMONARY HYPERTENSION IN RATS

Objective. To investigate the roles of apoptosis in the pulmonary artery remodeling of pulmonary hypertension secondary to hypoxia and illustrate the relative genes expression. Methods. Thirty rats were divided into hypoxia group（ 10％ O2, 8h/d） and normal control group. On the 15th day of hypoxia, pulmonary artery pressure and right ventricular hypertrophy index were measured and pulmonary artery vessels were studied by light microscope. Then terminal deoxynucleotidyl transferase- mediated dUTP nick- end labeling（ TUNEL） technique was used to detect nucleosomal DNA fragmentation of apoptotic cells. In situ hybridization and RT- PCR were used to detect the expression level of bcl- 2 and bax. Results. The pulmonary artery pressure and right ventricular hypertrophy index of hypoxia group were increased significantly, the pulmonary artery wall of hypoxic group become incrassate than control group. Apoptotic cells can be found in lung with hypoxia or without hypoxia. Compared with control group, apoptotic index of hypoxic group decreased significantly. Through the methods of in situ hybridization and RT- PCR, we found the expression of bcl- 2 increased whereas bax decreased significantly in the hypoxic group. Conclusion. The alternation in bcl- 2 and bax expression induced by hypoxia play an important role in the pulmonary artery remodeling which is the main pathologic change of pulmonary hypertension secondary to hypoxia.

Effects of NHE-1 ribozyme gene transfection on apoptosis of rat pulmonary artery smooth muscle cells in vitro

Objective : To investigate the effects of the transfection of NHE-1 ribozyme gene on the apoptosis of pulmonary artery smooth muscle cells (PASMC) in vitro. Methods: After NHE-1 ribozyme gene was designed, synthesized and then cloned into plasmid pLXSN, the recombined plasmid was tansfected into cultured rat PASMC. Expression of NHE-1 mRNA was detected with semi-quantitative RT-PCR. Intracellular pH (pHi) was measured by using fluorescence dye BCECF-AM. Cell cycle was measured with aid of flow cy-tometric DNA analysis. Cell apoptosis was observed with electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNED respectively. Results: The NHE-1 mRNA expression level and pHi value were significantly lower in PASMCs transfected with NHE-1 ribozyme gene than those transfected with pLXSN or without transfection. Meanwhile, the apoptosis rate of cells transfected with NHE-1 ribozyme gene was increased significantly. Morphology of cell apoptosis was observed in the cells transfected with NHE-1 ribozyme gene under an electron microscope. Conclusion: The transfection of NHE-1 ribozyme gene induces the apoptosis of PASMCs by inhibiting NHE-1 expression and intracellular acidification.

Intestinal ischemia and reperfusion induced second impairment of the rat heart

Objective: To study the changes of oxygen free radical, expression of apoptotic gene, ultrastructure of myocardial cell and second injury of the heart following the intestinal ischemia and reperfusion. Methods: Making the models of ischemia and reperfusion by clamping superior mesenteric artery, the concentration of NO and SOD in the blood was examined before clamping the artery and reperfusion for 0, 30, 60 min, 1, 3, and 7 d. The expression of Bax, Bal-2, and p53 in myocardial cell was studied by means of immunohistochemical SP method and the microstructure damage was observed under electron microscopy. Results: After clamping the superior mesenteric artery and reperfusion the concentration of NO increased continuously and reached a peak for reperfusion 7 d (P<0.01) but that of SOD decreased from 30 min to 7 d. The expression of Bax, p53 and Bcl-2 also increased obviously especially for reperfusion 30 min and 7 d following ischemia and reperfusion. After reperfusion for 30 min the positive cell rate of Bax, p53 and Bcl-2 was 53.6%, 45.9% and 67.9%, for reperfusion 7 d it was 52.4%, 43.4% and 31.9% respectively, but the positive cell rate of Bax and p53 was higher than that of Bcl-2. The result of electron microscopy observation showed mfofiliments breaked, dissolved and chromatin condensed. Conclusion: Intestinal ischemia and reperfusion of rat can induced the changes of oxygen free radical and the expression of apoptotic gene and second injury of myocardial cells.

pSVPoMcat modifying Schwann cell to protect injured spinal neurons in rats

Objective: To investigate the protective effect of pSVPoMcat (myelin basic protein microgene)modifying Schwann cell on injured spinal neurons. Methods: A model of rat spinal cord injured by hemisection was used. One hundred and twenty healthy SD rats of both sexes weighing 250 300 g were divided into three groups: Group A (n=40, treated with implantation of pSVPoMcat modifying Schwann cell), Group B (n= 40, treated with implantation of Schwann cell only) and Group C (n=400, treated with sham operation as the control). One week after operation the rat functional recovery was observed dynamically by using combined behavioral score (CBS) and cortical somatasensory evoked potentials, the spinal cord sections were stained by Nissl, acid phosphatase enzyme histochemistry and cell apoptosis was examined by methye green, terminal deoxynucleotidyl and the dUTP Nick end labeling technique. Quantitative analysis was done by computer image analysis system. Results: In Group A the injured neurons recovered well morphologically. The imaging analysis showed a result of Group A>Group B>Group C in the size of the neurons (P< 0.01 ). The percentage of ACP (acid phosphatase) stained area and the rate of apoptosis sequence were groups A<B<C. The change of tendency was correlated to their function recovery according to CBS. Conclusions: pSVPoMcat modifying Schwann cell implantation has protective effect on injured spinal neurons and promotes recovery of injured spinal cord function in rats.