子宫内膜腺上皮细胞中增殖与凋亡相关基因表达初探

目的:探讨不同类型子宫内膜腺上皮细胞的增殖和凋亡情况。方法:采用免疫组化和TUNEL方法检测不同类型子宫内膜组织中增殖细胞核抗原(PCNA)与细胞凋亡表达。结果:(1)从正常子宫内膜直至子宫内膜样癌的PCNA表达逐渐增加。在子宫内膜样腺癌中随着癌细胞分化程度的降低、肌层浸润深度的增加和手术病理分期的升高,PCNA表达逐渐增加,且各组之间PCNA表达均有显著性差异(P<0.05)(。2)从正常子宫内膜、子宫内膜增殖症到子宫内膜样腺癌细胞凋亡的表达逐渐增加。子宫内膜样腺癌中不同分化程度的癌细胞、不同手术病理分期之间凋亡表达均有显著差异(皆P<0.01);而不同肌层浸润深度之间凋亡表达无显著差异(P>0.05)。(3)PCNA表达与细胞凋亡在不典型增生、子宫内膜样癌中均呈负相关(分别r=-0.943,P<0.01;r=-0.976,P<0.01)。结论:PCNA是内膜细胞癌变过程中一项重要的指标,其在内膜癌的发生和发展中起重要作用,可作为估计患者预后的一个重要参数。细胞凋亡情况在一定程度上可以反映肿瘤的生物学行为。内膜细胞增殖与凋亡相互作用共同促进内膜细胞过度增殖,从而导致肿瘤的形成和进展。     

葛根素诱导血管平滑肌细胞凋亡的实验研究

目的：观察葛根素促进人脐动脉血管平滑肌细胞凋亡的作用，探讨葛根素抑制平滑肌细胞增殖的机制。方法：分离培养人脐动脉平滑肌细胞，不同浓度葛根素与细胞孵育，TUNEL检测葛根素诱导细胞凋亡，rt-PCR实验检测促凋亡基因Bax和抗凋亡基因Bcl-XL。结果：随着葛根素浓度升高，细胞生长受抑制，TUNEL阳性细胞显著增加。rt-PCR实验发现促凋亡基因Bax随药物浓度和作用时间的增加而上升，而且Bax、Bcl-XL基因表达比例有一定升高。结论：葛根素通过调节经典的Bax、Bcl-XL通路对血管平滑肌细胞凋亡有一定诱导作用。     

G-CSF/SCF exert beneficial effects via anti-apoptosis in rabbits with steroid-associated osteonecrosis

Objectives

Osteonecrosis is also known as avascular necrosis, and two types of cell death are included in the pathogenesis of osteonecrosis: necrosis and apoptosis. Apoptosis in the osteonecrosis of femoral head is thought to be the key determinant of glucocorticoid-induced cortical bone loss. The present study was implemented to evaluate the anti-apoptotic effect of Granulocyte colony-stimulating factor and stem cell factor (G-CSF/SCF) in rabbits with steroid-induced osteonecrosis.

Methods

In the experiment, osteonecrosis was induced by low-dose lipopolysaccharide and subsequent pulsed high-dose methylprednisolone. Rabbits in preventive medicine group were treated with100 μg/kg/d G-CSF and 25 μg/kg/d SCF. Then hematological and histomorphometric methods were used to investigate the treatment effects of osteonecrosis. Apoptosis was assessed via quantitative TUNEL staining and activated caspase-3 immunostaining and immunoblotting.

Results

The results showed that G-CSF/SCF treatment could increase the secretion of serum osteocalcin, but inhibit the expression of serum tartrate-resistant acid phosphatase (TRAP5b). The incidence of osteonecrosis was significantly decreased in Preventive group when compared with Steroid group (42.1% vs. 88.2%). Histomorphometric analysis showed that G-CSF/SCF pre-disposal treatment was able to increase trabecular mineral appositional rate (MAR) and bone formation rate (BFR). Quantitative TUNEL and activated caspase-3 levels showed lower apoptosis in the Preventive group.

Conclusions

In conclusion, G-CSF/SCF treatment could inhibit caspase-3-dependent apoptosis in osteocytes to exert beneficial effects in preventing steroid-induced ON in rabbit models.     

Apurinic/apyrimidinic endonuclease immunoreactivity in germ cells of experimental varicocele-induced rat testes

Increased germ cell apoptosis is related to oxidative DNA damage; therefore, we investigated whether there was a significant change in apurinic/apyrimidinic endonuclease (APE) in varicoceles. Experimental varicoceles were created by partial ligation of the left renal vein of adult male Sprague-Dawley rats, which were sacrificed at 1, 3 and 6 weeks after varicocele creation. Testicular tissues were sampled for TUNEL, Western blotting and immunohistochemistry. There was a significant increase in apoptotic germ cells in the ipsilateral testes 6 weeks after varicocele creation. Increased activation of p53, Bax and cleaved caspase-3 in the left testes was also noted. APE increased activation until 3 weeks after varicocele creation, and then decreased at 6 weeks after varicocele surgery. The spermatocytes were immunostained for both 8-hydroxy-2′-deoxyguanosine and APE, but the spermatogonia revealed only APE immunopositivity in the defective tubules. These results suggest that repression of APE is an underlying mechanism of augmented p53-dependent apoptosis in varicocele-induced rat testes and that remaining APE in the spermatogonia plays a decisive role in regaining testicular spermatogenic function after varicocelectomy.     

大鼠脊髓损伤后神经细胞凋亡及相关调控蛋白Caspase-3表达的时空分布

目的 观察大鼠脊髓损伤（SCI）后神经元细胞凋亡和相关调控蛋白Caspase-3表达的时间、空间分布规律。方法 SD大鼠50只,分为假手术组和SCI组,钳夹法建立SCI模型,用结晶紫（CV）染色法、TUNEL法和免疫组织化学方法观察SCI后6h、12h、24h、3d和7d损伤中心到头端0～5.00mm空间范围内脊髓神经细胞存活、凋亡以及相关调控蛋白Caspase-3的表达状况。 结果 CV染色发现,SCI后6h～7d在0～0.50mm空间范围内均未发现存活的神经细胞,在1.00～3.50mm距离内,存活的神经细胞数逐渐增加,4.00mm处达峰值;TUNEL结果发现,SCI后6h～7d在1.05～4.55mm范围内,神经细胞出现凋亡,3d时在2.55mm处,细胞凋亡达高峰,7d时显著减少;免疫组织化学结果发现,SCI后6h～7d在1.10～4.60mm范围内,神经元细胞出现Caspase-3阳性表达,3d在3.10mm处Caspase-3表达达到高峰,7d显著减少。 结论 SCI后,凋亡的神经细胞及其相关调控蛋白Caspase-3的表达有一定的时空分布规律。     

黄芪多糖对阿霉素诱导的心力衰竭模型大鼠的保护作用

目的 探讨黄芪多糖对大鼠阿霉素性心衰的保护作用。方法 取雄性SD大鼠30只,随机分为对照组、心衰模型组和黄芪多糖组,每组10只。黄芪多糖组连续14d黄芪多糖水溶液灌胃［3g/(kg·d)］,正常对照组和心衰模型组灌胃等量蒸馏水。心衰模型组和黄芪多糖组分4次静脉注射阿霉素［10.4mg/(kg·2d)］复制心衰模型。给药全部结束后,检测大鼠心肌重构、细胞凋亡和抗氧化性等指标。结果 阿霉素导致心肌纤维化,肌原纤维溶解断裂,线粒体肿胀变性,应用黄芩多糖能明显改善心衰症状。血流动力学和TUNEL染色进一步证实黄芪多糖能缓解阿霉素对心脏的损伤,这一作用可能是通过降低丙二醛(MDA)含量和Bax的表达,同时增加超氧化物歧化酶（SOD）活性和Bcl-2的表达来实现的。结论 黄芪多糖对阿霉素性心衰有较好的保护作用。     

Immunostimulatory DNA ameliorates experimental and spontaneous murine colitis

BACKGROUND & AIMS: Impaired mucosal barrier, cytokine imbalance, and dysregulated CD4(+) T cells play important roles in the pathogenesis of experimental colitis and human inflammatory bowel disease. Immunostimulatory DNA sequences (ISS-DNA) and their synthetic oligonucleotide analogs (ISS-ODNs) are derived from bacterial DNA, are potent activators of innate immunity at systemic and mucosal sites, and can rescue cells from death inflicted by different agents. We hypothesized that these combined effects of ISS-DNA could inhibit the damage to the colonic mucosa in chemically induced colitis and thereby limit subsequent intestinal inflammation. METHODS: The protective and the anti-inflammatory effect of ISS-ODN administration were assessed in dextran sodium sulfate-induced colitis and in 2 models of hapten-induced colitis in Balb/c mice. Similarly, these effects of ISS-ODN were assessed in spontaneous colitis occurring in IL-10 knockout mice. RESULTS: In all models of experimental and spontaneous colitis examined, ISS-ODN administration ameliorated clinical, biochemical, and histologic scores of colonic inflammation. ISS-ODN administration inhibited the induction of colonic proinflammatory cytokines and chemokines and suppressed the induction of colonic matrix metalloproteinases in both dextran sodium sulfate- and hapten-induced colitis. CONCLUSIONS: As the colon is continuously exposed to bacterial DNA, these findings suggest a physiologic, anti-inflammatory role for immunostimulatory DNA in the GI tract. Immunostimulatory DNA deserves further evaluation for the treatment of human inflammatory bowel disease.     

Glutathione-S-transferase P1-1 protects aberrant crypt foci from apoptosis induced by deoxycholic acid

BACKGROUND & AIMS: Aberrant crypt foci, precursors of colonic adenoma, are frequently positive for glutathione-S-transferase P1-1. Because deoxycholic acid is an apoptosis-inducing xenobiotic in the colon, we examined the possibility that aberrant crypt foci, through the cytoprotecting function of glutathione-S-transferase P1-1, resist deoxycholic acid-induced apoptosis, thereby surviving to become adenomas and subsequently cancer. METHODS: Glutathione-S-transferase P1-1 or cyclooxygenase-2 expression and the percentage of apoptotic cells in aberrant crypt foci were examined by immunohistochemistry and by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, respectively. Glutathione-S-transferase P1-1 was transfected into colon cancer cells (M7609) and human lung fibroblasts, and deoxycholic acid-induced apoptosis was evaluated by a dye-uptake assay and flow cytometry. Binding of deoxycholic acid to glutathione-S-transferase P1-1 was analyzed by circular dichroism and immunoprecipitation. Caspase activities were determined by colorimetric protease assay, and sulindac binding to glutathione-S-transferase P1-1 was determined by inhibition assay of glutathione-S-transferase P1-1 activity. RESULTS: Aberrant crypt foci showed positive immunostaining for glutathione-S-transferase P1-1 but negative staining for cyclooxygenase-2. The percentage of apoptotic cells in aberrant crypt foci was significantly lower than in healthy epithelium, and the difference became more apparent with deoxycholic acid treatment. The impaired sensitivity of aberrant crypt foci to deoxycholic acid was restored by the glutathione-S-transferase P1-1-specific inhibitor gamma-glutamyl-S-(benzyl)cysteinyl-R-phenylglycine diethylester. By transfection of glutathione-S-transferase P1-1, M7609 cells became more resistant to deoxycholic acid-induced apoptosis than mock transfectants. Direct binding of glutathione-S-transferase P1-1 to deoxycholic acid was proven by circular dichroism and by immunoprecipitation. The aberrant crypt foci in adenoma patients treated with sulindac, which was shown to bind to glutathione-S-transferase P1-1, underwent apoptosis in 4 days and mostly regressed in 2-3 months. CONCLUSIONS: Glutathione-S-transferase P1-1 protects aberrant crypt foci from deoxycholic acid-induced apoptosis and may play a pivotal role in early colon carcinogenesis.     

The effects of water immersion and restraint stress on the expressions of apelin,apelin receptor (APJR) and apoptosis rate in the rat heart

Apelin has been identified as an endogenous ligand of the orphan G-protein-coupled apelin receptor (APJR). These receptors are widely expressed in the central nervous system and periphery and play a role in the regulation of fluid and glucose homeostasis, feeding behavior, vessel formation, cell proliferation and immunity. We aimed to investigate whether water immersion and restraint stress have effects on apelin and APJR expression and apoptosis in heart tissue of male Wistar rats. The cardiac tissues were obtained from control, water immersion and restraint stress (WIRS) and apelin antagonist (F13A) + WIRS groups of rats and embedded in paraffin wax. Immunohistochemical staining methods were used to localize apelin, APJR and TUNEL immunopositive cells. H-SCORE was used for semi-quantitative determinations. Apelin protein levels were determined by Western blot in the cardiac tissues and plasma corticosteroid levels were measured by enzyme immunoassay (EIA). Apelin immunolocalization was found especially in endothelial cells and mast cells and faintly in cardiomyocytes, APJR immunostaining was shown in endothelial cells and cardiomyocytes, and TUNEL reaction was observed in endothelial cells and in some fibroblasts. Apelin expression was significantly increased in the WIRS and F13A + WIRS groups compared to the control group. The APJR reaction was similar in all groups. The number of TUNEL-positive cells was significantly higher in the F13A + WIRS group than that of the control group. Our study showed that WIRS for 6 h increased plasma corticosterone levels and cardiac apelin expression in rats. The increased levels of apelin inhibited stress-induced apoptosis in heart. These results may be important for the therapeutic approach to a variety of stress-related heart disease.