db/db自发性糖尿病小鼠睾丸生殖细胞凋亡及相关基因Bcl-2和Bax表达

目的　研究糖尿病时生殖细胞凋亡及相关基因Bcl 2和Bax表达变化。方法　以脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记法 (TUNEL)检测生殖细胞的凋亡 ,免疫组化ABC法检测凋亡相关基因Bcl 2和Bax的表达变化。结果　凋亡管数及凋亡阳性率糖尿病组均较正常高 ,而且随糖尿病病程进展呈明显增加趋势 ,对照组中随加龄二者也有增加。Bcl 2蛋白以散在的颗粒状大量分布于除精原细胞的各级生精细胞中 ,且以精子细胞质为最多。随糖尿病病变加重 ,Bcl 2表达减少 ,而且 ,糖尿病组比对照组Bcl 2表达率低。Bax蛋白比Bcl 2蛋白表达量少 ,以散在的颗粒状分布于曲细精管基底部的精原细胞和支持细胞中。随糖尿病病程进展和年龄增加 ,表达均呈增加趋势 ,而且 ,糖尿病组比对照组Bax表达率高。结论　在db/db自发性糖尿病小鼠睾丸 ,伴随生殖细胞凋亡增加 ,Bcl 2表达减少 ,Bax表达增加 ,凋亡及相关基因在糖尿病生殖功能障碍中起重要作用     

db／db自发性糖尿病小鼠睾丸EGFR，c—Fos表达研究

目的：研究表皮生长因子受体（Epithelium growth factor receptor EGFR)和原癌基因c-Fos在db/db(diabetes obese)自发性糖尿病小鼠睾丸生殖细胞伯表达变化。方法：免疫组化ABC法。结果：EGFR在精母细胞。精子细胞、支持细胞和间质细胞均呈阳性反应。c-Fos免疫阳性细胞主要为精母细胞,圆形精子细胞和变形期精子,间质血管内皮细胞也有强阳性表达,另外,少数支持细胞,管周肌样细胞也有着色,以上两组指标。随糖尿病病程进展（糖尿病组）及年龄增加（对照组）,其阳性率呈现明显下降趋势,并且每年龄段糖尿病组阳性率明显低于相应正常对照组。结论：糖尿病睾丸中EGFR的减少例EGF与受体结合减少,第二信使cAMP和“第三信使”c-Fos均减少,以致核转录和核基因表达减弱,最终表现为糖尿病生殖细胞增殖减弱,生殖功能障碍。     

凋亡细胞在db/db自发性糖尿病小鼠颌下腺的分布

目的:观察凋亡细胞在db/db糖尿病小鼠颌下腺中的分布。方法:选取3、4、6、8、10月龄db/db糖尿病小鼠及相应月龄的dh/m~(?)小鼠颌下腺,应用TUNEL标记方法染色后进行图像分析.统计凋亡细胞在颌下腺组织中分布的细胞阳性率。结果:随着糖尿病的发展,颌下腺组织出现腺体萎缩及颗粒曲管数目减少,实质细胞排列不整齐,呈簇状堆集,纤维及血管增多。凋亡细胞在对照组及糖尿病组颌下腺中均有分布,糖尿病组凋亡细胞阳性率高于对照组。糖尿病组与对照组凋亡细胞阳性率随月龄增大均呈增加趋势。结论:db/db糖尿病可导致颌下腺组织萎缩及实质细胞形态学改变;凋亡细胞阳性率在糖尿病组随疾病发展而增加显著。这与糖尿病腺体萎缩和功能受损相一致。     

增龄对db/db糖尿病小鼠睾丸PCNA表达的影响

增殖细胞核抗原 (PCNA) ,又称细胞周期蛋白 (cylin) ,是一种与细胞周期相关的 36 k D酸性核蛋白 ,作为 DNA聚合酶δ的一个辅助因子发挥作用 ,为 DNA 复制起始所必需。C5 7BL/Ks J- db/db小鼠是第 4号染色体恶性突变所致的一种单基因遗传自然发病糖尿病小鼠 ,是研究人类 型 (非胰岛素依赖型 )糖尿病的良好动物模型。本研究应用免疫组化方法探讨糖尿病和增龄对雄性不育 db/db小鼠睾丸组织PCNA表达的影响。实验选用 8周 ,12周 ,2 0周 ,32周糖尿病和正常小鼠 ,4%多聚甲醛经左心室灌注固定 ,取左侧睾丸 ,Bouin液后固定 ,常规石蜡包埋切…     

饮酒对小鼠睾丸生精小管一氧化氮合酶、增殖细胞核抗原表达及细胞凋亡影响的研究

目的　探讨酒精对小鼠睾丸的组织结构、内皮型一氧化氮合酶 (eNOS)、增殖细胞核抗原 (PCNA)及细胞凋亡的影响。　方法　用 5 %、10 %及 15 % 3种不同浓度的酒精作用于 2 2d龄小鼠 ,取睾丸做石蜡切片、HE染色 ;用免疫组织化学方法检测睾丸eNOS、细胞增殖的变化 ;TUNEL法检测细胞凋亡的变化 ,并进行统计学分析。　结果　随着酒精浓度的增大 ,睾丸组织结构发生明显改变 ,生精小管的直径逐渐减小 ,eNOS阳性细胞面积密度逐渐增大 ,单位面积内PCNA阳性细胞和凋亡细胞数目增加 ,高浓度酒精组与其他组差异极显著 (P <0 0 1)。　结论　酒精可使生精小管的直径变小 ,eNOS及PCNA表达增强 ,凋亡细胞增加并随酒精浓度的增大而变化加重。这可能是过量饮酒导致生精细胞减少 ,生殖能力降低的重要因素。     

DahlS高血压大鼠生精细胞凋亡及Bcl-2蛋白的表达

目的　为阐明高血压导致生精障碍的确切机制。方法　应用原位末端标记法 (TUNEL)、免疫组织化学、流式细胞检测及电镜等方法 ,对DahlS高血压大鼠生精细胞凋亡的定性、定位、定量及其相关蛋白Bcl 2的表达进行了研究。结果　实验组随高血压病程延长 ,血压升高 ,生精细胞凋亡率增高 ,15、17周龄明显高于对照组 (P <0 0 5 )。Bcl 2蛋白表达降低 ,15、17周龄与对照组相比差异显著 (P <0 0 5 )。随糖尿病病变加重 ,Bcl 2表达减少 ,而且 ,糖尿病组比对照组Bcl 2表达率低。随糖尿病病程进展和年龄增加 ,表达均呈增加趋势 ,而且 ,糖尿病组比对照组Bax表达率高。结论　高血压可导致凋亡抑制基因Bcl 2表达降低 ,从而使生精细胞凋亡增加 ,这可能是高血压导致生精障碍的机制之一     

糖尿病小鼠胰岛β细胞结构的光镜和电镜研究

目的观察2型糖尿病模型db/db小鼠胰岛β细胞的超微结构、胰岛素表达及数量变化,探讨β细胞的病理改变与2型糖尿病病因的关系。方法分别选取3、5、8月龄尾静脉空腹血糖高于10.1mmol/L,且肥胖的db/db自发性糖尿病小鼠,每组8只,作为糖尿病组;选取相应年龄段尾静脉空腹血糖低于6.0mmol/L,体重正常的db/+m表型正常小鼠,每组8只,作为对照组。于相应年龄段取胰尾,用于透射电镜观察、免疫组织化学观察和图像分析。结果电镜下随病情进展,db/db小鼠胰岛β细胞内的分泌颗粒数量明显减少,有的细胞甚至缺如,致密芯电子密度降低,β细胞可见凋亡的早期改变以及细胞核和细胞器的病理改变,细胞间髓样小体增多。免疫组织化学显示同月龄糖尿病组小鼠胰岛β细胞阳性率和胰岛素蛋白平均光密度值(OD值)低于相应对照组(p<0.05),且随着病程的进展,db/db小鼠胰岛β细胞阳性率和胰岛素表达呈现递减趋势(p<0.05)。结论2型糖尿病β细胞的超微结构遭到破坏,引起β细胞合成分泌胰岛素障碍和数量减少,与2型糖尿病病情的轻重有关,反映了2型糖尿病病程不同阶段的病机特点。     

小鼠胰岛内分泌细胞的构成及其与Ⅱ型糖尿病的关系

目的观察和分析II型糖尿病动物模型db/db小鼠病程进展中胰岛内分泌细胞的构成变化及与II型糖尿病病因的关系。方法分别选取3、5、8、10和12月龄的尾静脉空腹血糖高于10.0mmol/L且肥胖的db/db自发性糖尿病小鼠,每组5只,作为糖尿病组;选取相应年龄段的尾静脉空腹血糖低于6.0mmol/L体重正常的db/+m表型正常小鼠,每组5只,作为对照组。于相应年龄段取胰尾,用于免疫组织化学观察和比较。结果各月龄糖尿病组胰岛B细胞(胰岛素阳性细胞)阳性率低于对照组(p<0.05),而A细胞(胰高血糖素阳性细胞)和D细胞(生长抑素阳性细胞)阳性率分别高于对照组(p<0.05)。糖尿病组胰岛B细胞阳性率随自发性db/db糖尿病小鼠病程进展呈降低趋势(p<0.05),而A细胞和D细胞阳性率则呈增高趋势(p<0.05);对照组3种细胞阳性率无变化(P>0.05)。结论II型糖尿病动物模型db/db小鼠胰岛内分泌细胞的构成变化与糖尿病病情的轻重有关,胰岛内分泌细胞之间的比例失衡可能与II型糖尿病的发病有关。     

小鼠胰岛内分泌细胞的构成及其与‖型糖尿病的关系

目的 观察和分析Ⅱ型糖尿病动物模型db/db小鼠病程进展中胰岛内分泌细胞的构成变化及与Ⅱ型糖尿病病因的关系.方法 分别选取3、5、8、10和12月龄的尾静脉空腹血糖高于10.0mmol/L且肥胖的db/db自发性糖尿病小鼠.每组5只,作为糖尿病组;选取相应年龄段的尾静脉空腹血糖低于6.0mmol/L体重正常的db/+m表型正常小鼠,每组5只,作为对照组.于相应年龄段取胰尾,用于免疫组织化学观察和比较.结果 各月龄糖尿病组胰岛B细胞(胰岛素阳性细胞)阳性率低于对照组(p＜0.05),而A细胞(胰高血糖素阳性细胞)和D细胞(生长抑素附性细胞)阳性率分别高于对照组(p＜0.05).糖尿病组胰岛B细胞阳性率随自发性db/db糖尿病小鼠病程进展旱降低趋势(p＜0.05),而A细胞和D细胞阳性率则旱增高趋势(p＜0.05);对照组3种细胞阳性率无变化(P＞0.05).结论 Ⅱ型糖尿病动物模型db/db小鼠胰岛内分泌细胞的构成变化与糖尿病病情的轻重有关,胰岛内分泌细胞之间的比例失衡可能与Ⅱ型糖尿病的发病有关.     

BID蛋白促细胞凋亡增殖失衡作用与IgA肾病患者病情的关系

目的 探讨IgA肾病(IgAN)不同病变程度肾组织中BID蛋白的表达与细胞凋亡、增殖的关系。方法 免疫组化法检测肾组织PCNA、BID蛋白和FN,用原位末端标记法(TUNEL)结合光镜形态学检测细胞凋亡情况。结果 随IgAN病变程度加重，BID蛋白、PCNA、凋亡细胞及FN表达逐渐增强(P<0.05), Ⅳ级最强 (PCNA：Ⅲ、Ⅳ相当)；肾小球内BID蛋白与凋亡细胞比值、PCNA 、FN表达,肾小球硬化率及尿蛋白排泄量正相关，与eGFR负相关(Ｐ均<0.01)。结论 BID蛋白可通过促进细胞凋亡增殖失平衡促进IgAN病程进展，可能为判断预后的一项重要病理指标。     

Proliferation and apoptotic rates and increased frequency of p63‐positive cells in the prostate acinar epithelium of alloxan‐induced diabetic rats

The effects of experimental type 1 diabetes were investigated in the acinar epithelium of rat ventral prostate, focusing on the rates of cell proliferation and the frequency of apoptosis and p63‐positive cells. Type 1 diabetes was induced in adult male Wistar rats by a single alloxan administration (42 mg/kg b.w.) and its effects were analysed for 1 week and 3 months after the establishment of the disease. A group of diabetic rats was treated daily with 5 IU of insulin during 1 week after diabetes had been diagnosed. Immunocytochemical methods for the localization of cell proliferation antigen (PCNA), androgen receptor (AR) and p63 protein were carried out, and apoptotic cells were identified by TUNEL essay. In diabetic rats, testosterone levels reduced drastically after 1 week and in a lower degree after 3 months. In short‐term diabetic rats, cell proliferation decreased, and in medium‐term, epithelial apoptotic rates increased. In both periods after the onset of diabetes, the frequency of p63‐positive cells doubled. Insulin treatment was effective in preventing testosterone decrease, p63‐positive cell increase and apoptotic rates, but did not interfere in cell proliferation. This investigation shows that, soon after diabetes onset, there are important modifications in cell proliferation within the acinar prostatic epithelium, and in longer term, there is a marked impact on kinetics of differentiation and cell death, which may initially be attributable to an androgenic fall, but is probably also because of other factors related to diabetes, as changes are considerably different from those resulting from castration.     

Cell death and cell proliferation in mouse submandibular gland during early post-irradiation phase

The effects of irradiation on different cell compartments in the submandibular gland were analyzed in adult C57BL/6 mice exposed to X-ray irradiation and followed up for 10 days. Apoptosis was quantified using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling method (TUNEL). Cell proliferation was detected using immunohistochemistry for proliferating cell nuclear antigen (PCNA). Radiation-induced apoptosis occurred rapidly, reaching a maximum 3 days post-irradiation. The percentage of apoptotic cells increased with the irradiation dose. At day 1 post-irradiation, cell proliferation was significantly reduced in comparison to sham-irradiated controls. After post-irradiation arrest of the cell cycle, proliferation increased in all gland compartments, reaching a maximum at day 6 post-irradiation. The proliferation response corresponded to the dose of irradiation. We suggest that the reason for gland dysfunction could be the coexistence of high apoptotic and proliferative activity in the irradiated gland.     

Renal distal tubule proliferation and increased aquaporin 2 level but decreased urine osmolality in db/db mouse: treatment with chromium picolinate

Hallmark features of type 2 diabetes mellitus include glucosuria and polyuria. Further, renal aquaporin 2 is pivotal to regulation of fluid excretion and urine osmolality. Accordingly, we tested the hypothesis that the db/db mouse displays increased glucosuria and fluid excretion but reduced urine osmolality in association with decreased renal aquaporin 2 level. In addition, we examined the effect of chromium picolinate (Cr(pic)3) which is purported to improve glycemic control. The db/db mice excreted more urine in association with marked glucose excretion but lower urine osmolality than db/m control group. Light microscopic examination of renal tissue revealed proliferation of tubular structures in db/db compared to the db/m mice, a feature validated with Ki67 immunostaining. Further, these tubules showed generally similar immunostaining intensity and pattern for aquaporin 2 indicating that proliferated tubules are of distal origin. On the other hand, renal aquaporin 2 protein level was significantly higher in the db/db than db/m group. Treatment of db/db mice with Cr(pic)3 reduced plasma glucose and hemoglobin A1c (~15-17%, p<0.05) and Ki67 positive cells but other parameters were similar to their untreated counterparts. Collectively, these findings suggest that proliferation of renal distal tubules and increased aquaporin 2 level likely represent an adaptive mechanism to regulate fluid excretion to prevent dehydration in the setting of marked glucosuria in the db/db mouse, features not affected by Cr(pic)3 treatment. These observations are of relevance to increasing interest in developing therapeutic agents that facilitate renal glucose elimination.     

In vivo collagen metabolism in spontaneously diabetic (db/db) mice

To further define the pathogenesis of diabetic connective tissue lesions, collagen synthesis and degradation were measured in vivo in spontaneously diabetic db/db mice. A double isotopic labeling technique, in which 14C-labeled and 3H-labeled proline were injected into the same mouse 7 days apart, was applied. Collagen synthesis and degradation were assessed in skins, intestines, hearts, and kidneys. There were no changes in collagen metabolism in the intestines of the diabetic mice. In all other tissues, collagen degradation was accelerated. Collagen synthesis was decreased in skins, but increased in the hearts and kidneys of the diabetic mice. These tissue-specific changes in collagen metabolism resulted in a net loss of collagen in all tissues examined except intestines. The results of this study provide insight into the mechanisms leading to connective tissue defects occurring in diabetes mellitus.     

Gonadal steroid modulation of the diabetes (db/db) mutation-induced hyperlipometabolic, hypogonadal syndrome: restoration of female reproductive tract cytochemical and structural indices.

The gonadal steroids, 17-B-estradiol (E2) and progesterone (P), are recognized to stimulate cellular gluco- and lipo-metabolic compensatory cascades which counteract the deleterious influences of the diabetes (db/db) mutation (i.e., leptin membrane receptor defect) which promotes a progressive, hypercytolipidemia-induced premature involution of the female reproductive tract (FRT). The current studies define the therapeutic efficacy of E2 (1 microg/3.5 days) and P (1 mg/3.5 days) treatments (HRx) on utero-ovarian structural and cytochemical (gluco-/lipo-metabolic) maintenance, and the prevention of premature nuclear apoptosis and cytostructural disruption, following the expression of progressive db/db-induced hypercytolipidemia. Control (normal: +/+ and +/?) and diabetes (db/db) genotype groups of 8-week-old (i.e., overt phase of the db/db-hypogonadal syndrome) C57BL/KsJ mice were prepared for high resolution (HRLM) cytochemical and transmission electron (TEM) microscopic analysis of cytolipidemia and nuclear apoptosis (TUNEL-labeled 3'-DNA fragmentation) indices from uterine and ovarian secondary (early antral) follicular tissue samples. Compared to HRx controls, the db/db mutation induced a dramatic increase in cytolipid vacuole volume and density within all ovarian follicular granulosa cells (GC) and uterine endometrial epithelial (UEE) layers. The co-localization of nuclear apoptotic 3'-DNA fragments within identified hyperlipidemic granulosa cells was coincident with the cytochemical and ultrastructural identification of lipid penetration through the nuclear envelope in db/db mutants. P-HRx moderated the severity of db/db-induced GC and UEE hypercytolipidemia, reducing the cytodensity of lipid vacuole accumulations and maintaining cytoplasmic organelle structure, organization, and nuclear membrane integrity. In contrast, E2-HRx resulted in a dramatic reduction in db/db cytolipidemia in both ovarian GC and UEE tissue compartments. Following E2-HRx, UEE cells demonstrated non-pycnotic nuclear profiles, reduced nuclear apoptosis TUNEL-labeling, increased cytoplasmic organelle density profiles and a pronounced cytoplasmic cisternal expansion indicative of active cellular nutrient/metabolite trafficking. Ovarian follicular GC populations demonstrated minimal cytolipidemia, a restored cytoarchitecture with prominent organelle compartments and reduced TUNEL-indexed nuclear lipoapoptosis. These results are the first cytochemical and ultrastructural indications that P- and E2-HRx compensate for the genetic db/db mutation-induced metabolic disturbances, which promote utero-ovarian hypercytolipidemia and the coincident nuclear lipoapoptosis culminating in the expressed diabetes hypogonadal syndrome. The capability of P-HRx to moderate the severity of utero-ovarian involution in db/db mutants, and of E2-HRx to restore and maintain viable GC and UEE cyto-chemical and -structural indices under normoglycemic conditions, suggests that chronic, low-dose cyclic P- and E2-HRx stimulate cellular gluco- and lipo-metabolic cascades which compensate for the lack of leptin signaling in these single-gene, obese-Type II diabetic mutants. The compensatory endometabolic maintenance of utero-ovarian cellular and nuclear architecture suggests that the gluco- and lipo-metabolic disregulation may be therapeutically prevented or reversed, restoring reproductive tract cytointegrity and function, reducing the manifestation of hypogonadal reproductive sterility and db/db compromise of the female reproductive tract.     

Feeding, activity, and body temperature following 6-hydroxydopamine lesions in diabetes (db/db) mice

Reductions in central catecholamines produced by intraventricular injections of 6-hydroxydopamine (6-OHDA) cause weight loss and decreased plasma glucose in diabetes (db/db) mice. The effects of this treatment were examined in short-term (64-day) and long-term (120-day) survival groups. Diabetes mice treated with 6-OHDA decreased food intake, lost weight, and maintained a lower weight than vehicle-treated controls until vehicle-treated animals began to enter the terminal stages of the syndrome, indicated by a loss of body weight. Diabetes mice given 6-OHDA lost weight despite reduced body temperatures and activity levels. Blood glucose levels were always lower in 6-OHDA than in ad lib fed vehicle-treated db/db mice. The 6-OHDA treatment also improved pancreatic islet granulation. Pair feeding vehicle-treated with 6-OHDA-treated db/db mice did not halt weight gain in the vehicle-treated group. However, measurement of carcass fat indicated similar losses in db/db-6-OHDA mice and vehicle-treated mice when the vehicle group was pair-fed with lean controls. Treatment with 6-OHDA produced long-term improvement in the diabetes syndrome, but the decreased body weight of the 6-OHDA-treated diabetes mice could not be completely accounted for by changes in food intake or measured indices of energy expenditure.