精索静脉曲张大鼠睾丸自噬对生精细胞的影响

目的:研究精索静脉曲张(VC)大鼠模型中睾丸不同水平自噬对生精细胞凋亡的影响。方法:雄性SD大鼠54只随机分为6组:空白对照组、雷帕霉素对照组、氯喹对照组各6只,VC组、VC+雷帕霉素组、VC+氯喹组各12只。采用HE染色观察睾丸、附睾组织形态学变化,并对睾丸及附睾中精子形成情况进行评分,TUNEL染色检测生精细胞凋亡指数(AI),Western印迹检测LC3、p62、Bax、Bcl-2的表达。结果:空白对照组、雷帕霉素对照组、氯喹对照组大鼠睾丸及附睾组织均未发生明显形态学变化,精子形成情况评分及AI亦无显著差异(P>0.05);VC组大鼠睾丸及附睾组织发生明显病理损伤,精子形成情况评分显著降低(P<0.01),AI显著升高(P<0.01),但VC+雷帕霉素组较VC组明显改善,而VC+氯喹组较VC组轻度加重。此外,与空白对照组比较,VC组自噬相关蛋白LC3(包括LC3-Ⅱ/LC3-Ⅰ的比值)和促凋亡蛋白Bax表达显著增加(P<0.01),抑凋亡蛋白Bcl-2表达则显著降低(P<0.01);且VC+雷帕霉素组LC3与Bcl-2表达显著高于VC组(P<0.01),p62和Bax表达则显著低于VC组(P<0.01);而VC+氯喹组LC3与Bcl-2表达显著低于VC组(P<0.01),p62和Bax的表达则显著高于VC组(P<0.01)。结论:VC可诱导大鼠睾丸自噬和生精细胞凋亡,上调自噬可抑制生精细胞凋亡,阻滞自噬则可促进生精细胞凋亡。     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

目的 探讨雄激素剥夺条件下阻断自噬后LNCaP细胞凋亡变化与半胱天冬酶(caspase)激活的关系.方法 应用激光共聚焦显微镜、RT-PCR方法观察雄激素剥夺致细胞自噬增加后,利用DAPI染色观察细胞凋亡变化及药物抑制caspase后对凋亡的影响.结果 ①雄激素去除后LNCaP细胞自噬体增加,标准培养基(CM)培养下LNCaP细胞自噬体数量为1.90分;无血清培养基(SF)中细胞自噬体数量增高为2.64分;加入双氧睾酮(SFA组)后细胞自噬体下降至1.85分(P<0.01).CM中LNCaP细胞LC3 mRNA表达率为23%,血清饥饿12 h后,LC3表达量上调至100%,而SFA组LC3 mRNA表达量为86%;血清饥饿24 h后,SF组LC3 mRNA表达量为62%,SFA组为35%.②SF组和SFA组LNCaP细胞基础凋亡率分别为(3.19±1.09)0A和(3.01±0.33)%,加入3-甲基腺嘌呤(3-MA)阻断自噬24 h后,SF组凋亡率为(10.90±2.91)%,SFA组为(4.63±1.69)%.SF+3-MA组中加入Z-VAD-FMK后,细胞凋亡减至(1.16±0.52)%.组间差异有统计学意义(P<0.01).结论 剥夺雄激素后LNCaP细胞中自噬明显增加,阻断自噬后凋亡发生率增加.     

Necrostatin-1对压力诱导的髓核细胞自噬及凋亡的影响

目的:观察Necrostatin-1对压力诱导的大鼠髓核细胞自噬及凋亡的影响。方法:从3月龄大鼠腰段脊柱提取髓核细胞,选取第2代细胞进行实验,分为对照(生理盐水)组和Necrostatin-1组,1.0 Mpa压力条件下培养0、24、36 h。单丹黄酰尸胺(MDC)染色荧光显微镜观察自噬泡及细胞膜上MDC荧光表达情况,...     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate GFP-LC3 structures were accumulated in the cells cultured in serum-free medi-um (SF)(2.64 scores). In contrast, the number of punctate GFP-LC3 remained at a very low level (1.85 scores), when cells were incubated with DHT in SFA(serum-free medium+DHT). Statistical analysis showed the significant difference between SF and SFA (P<0.01). Semiquantitative RT-PCR was employed to examine the mRNA expression of LC3. Indeed, cells grown in the medium without serum had a higher LC3 mRNA expression with the highest at 12 hour time point as compared with the cells grown in CM. DHT treatment reduced the level of LC3 mRNA. ②Blockage of autophagy by 3-MA increased the apoptosis of LNCaP cells. LNCaP cells in SF and SFA just presented a basal level of apoptosis, which is (3.19±1.09)% and (3.01±0.33)% , respectively. Under androgen-free con-ditions, inhibition of autophagy by 3-MA could increase apoptosis significantly(10. 90±2.91%). While Z-VAD-FMK, a pan Caspase inhibitor, was able to suppress this apoptotic process to the level of (1.16±0.52)%, which was statistically significant(P<0.01). Conclusions Androgen removal can lead to the increase of autophagy in LNCaP cells. Moreover, inhibition of autophagy promotes the occurrence of apoptosis.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate GFP-LC3 structures were accumulated in the cells cultured in serum-free medi-um (SF)(2.64 scores). In contrast, the number of punctate GFP-LC3 remained at a very low level (1.85 scores), when cells were incubated with DHT in SFA(serum-free medium+DHT). Statistical analysis showed the significant difference between SF and SFA (P<0.01). Semiquantitative RT-PCR was employed to examine the mRNA expression of LC3. Indeed, cells grown in the medium without serum had a higher LC3 mRNA expression with the highest at 12 hour time point as compared with the cells grown in CM. DHT treatment reduced the level of LC3 mRNA. ②Blockage of autophagy by 3-MA increased the apoptosis of LNCaP cells. LNCaP cells in SF and SFA just presented a basal level of apoptosis, which is (3.19±1.09)% and (3.01±0.33)% , respectively. Under androgen-free con-ditions, inhibition of autophagy by 3-MA could increase apoptosis significantly(10. 90±2.91%). While Z-VAD-FMK, a pan Caspase inhibitor, was able to suppress this apoptotic process to the level of (1.16±0.52)%, which was statistically significant(P<0.01). Conclusions Androgen removal can lead to the increase of autophagy in LNCaP cells. Moreover, inhibition of autophagy promotes the occurrence of apoptosis.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate GFP-LC3 structures were accumulated in the cells cultured in serum-free medi-um (SF)(2.64 scores). In contrast, the number of punctate GFP-LC3 remained at a very low level (1.85 scores), when cells were incubated with DHT in SFA(serum-free medium+DHT). Statistical analysis showed the significant difference between SF and SFA (P<0.01). Semiquantitative RT-PCR was employed to examine the mRNA expression of LC3. Indeed, cells grown in the medium without serum had a higher LC3 mRNA expression with the highest at 12 hour time point as compared with the cells grown in CM. DHT treatment reduced the level of LC3 mRNA. ②Blockage of autophagy by 3-MA increased the apoptosis of LNCaP cells. LNCaP cells in SF and SFA just presented a basal level of apoptosis, which is (3.19±1.09)% and (3.01±0.33)% , respectively. Under androgen-free con-ditions, inhibition of autophagy by 3-MA could increase apoptosis significantly(10. 90±2.91%). While Z-VAD-FMK, a pan Caspase inhibitor, was able to suppress this apoptotic process to the level of (1.16±0.52)%, which was statistically significant(P<0.01). Conclusions Androgen removal can lead to the increase of autophagy in LNCaP cells. Moreover, inhibition of autophagy promotes the occurrence of apoptosis.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate GFP-LC3 structures were accumulated in the cells cultured in serum-free medi-um (SF)(2.64 scores). In contrast, the number of punctate GFP-LC3 remained at a very low level (1.85 scores), when cells were incubated with DHT in SFA(serum-free medium+DHT). Statistical analysis showed the significant difference between SF and SFA (P<0.01). Semiquantitative RT-PCR was employed to examine the mRNA expression of LC3. Indeed, cells grown in the medium without serum had a higher LC3 mRNA expression with the highest at 12 hour time point as compared with the cells grown in CM. DHT treatment reduced the level of LC3 mRNA. ②Blockage of autophagy by 3-MA increased the apoptosis of LNCaP cells. LNCaP cells in SF and SFA just presented a basal level of apoptosis, which is (3.19±1.09)% and (3.01±0.33)% , respectively. Under androgen-free con-ditions, inhibition of autophagy by 3-MA could increase apoptosis significantly(10. 90±2.91%). While Z-VAD-FMK, a pan Caspase inhibitor, was able to suppress this apoptotic process to the level of (1.16±0.52)%, which was statistically significant(P<0.01). Conclusions Androgen removal can lead to the increase of autophagy in LNCaP cells. Moreover, inhibition of autophagy promotes the occurrence of apoptosis.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate GFP-LC3 structures were accumulated in the cells cultured in serum-free medi-um (SF)(2.64 scores). In contrast, the number of punctate GFP-LC3 remained at a very low level (1.85 scores), when cells were incubated with DHT in SFA(serum-free medium+DHT). Statistical analysis showed the significant difference between SF and SFA (P<0.01). Semiquantitative RT-PCR was employed to examine the mRNA expression of LC3. Indeed, cells grown in the medium without serum had a higher LC3 mRNA expression with the highest at 12 hour time point as compared with the cells grown in CM. DHT treatment reduced the level of LC3 mRNA. ②Blockage of autophagy by 3-MA increased the apoptosis of LNCaP cells. LNCaP cells in SF and SFA just presented a basal level of apoptosis, which is (3.19±1.09)% and (3.01±0.33)% , respectively. Under androgen-free con-ditions, inhibition of autophagy by 3-MA could increase apoptosis significantly(10. 90±2.91%). While Z-VAD-FMK, a pan Caspase inhibitor, was able to suppress this apoptotic process to the level of (1.16±0.52)%, which was statistically significant(P<0.01). Conclusions Androgen removal can lead to the increase of autophagy in LNCaP cells. Moreover, inhibition of autophagy promotes the occurrence of apoptosis.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate GFP-LC3 structures were accumulated in the cells cultured in serum-free medi-um (SF)(2.64 scores). In contrast, the number of punctate GFP-LC3 remained at a very low level (1.85 scores), when cells were incubated with DHT in SFA(serum-free medium+DHT). Statistical analysis showed the significant difference between SF and SFA (P<0.01). Semiquantitative RT-PCR was employed to examine the mRNA expression of LC3. Indeed, cells grown in the medium without serum had a higher LC3 mRNA expression with the highest at 12 hour time point as compared with the cells grown in CM. DHT treatment reduced the level of LC3 mRNA. ②Blockage of autophagy by 3-MA increased the apoptosis of LNCaP cells. LNCaP cells in SF and SFA just presented a basal level of apoptosis, which is (3.19±1.09)% and (3.01±0.33)% , respectively. Under androgen-free con-ditions, inhibition of autophagy by 3-MA could increase apoptosis significantly(10. 90±2.91%). While Z-VAD-FMK, a pan Caspase inhibitor, was able to suppress this apoptotic process to the level of (1.16±0.52)%, which was statistically significant(P<0.01). Conclusions Androgen removal can lead to the increase of autophagy in LNCaP cells. Moreover, inhibition of autophagy promotes the occurrence of apoptosis.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate GFP-LC3 structures were accumulated in the cells cultured in serum-free medi-um (SF)(2.64 scores). In contrast, the number of punctate GFP-LC3 remained at a very low level (1.85 scores), when cells were incubated with DHT in SFA(serum-free medium+DHT). Statistical analysis showed the significant difference between SF and SFA (P<0.01). Semiquantitative RT-PCR was employed to examine the mRNA expression of LC3. Indeed, cells grown in the medium without serum had a higher LC3 mRNA expression with the highest at 12 hour time point as compared with the cells grown in CM. DHT treatment reduced the level of LC3 mRNA. ②Blockage of autophagy by 3-MA increased the apoptosis of LNCaP cells. LNCaP cells in SF and SFA just presented a basal level of apoptosis, which is (3.19±1.09)% and (3.01±0.33)% , respectively. Under androgen-free con-ditions, inhibition of autophagy by 3-MA could increase apoptosis significantly(10. 90±2.91%). While Z-VAD-FMK, a pan Caspase inhibitor, was able to suppress this apoptotic process to the level of (1.16±0.52)%, which was statistically significant(P<0.01). Conclusions Androgen removal can lead to the increase of autophagy in LNCaP cells. Moreover, inhibition of autophagy promotes the occurrence of apoptosis.     

硫酸糖基化蛋白2在去势大鼠前列腺中的表达

目的 探讨硫酸糖基化蛋白2（SGP－2）在去势大腹叶前列腺表达的规律及其与前列腺凋亡的相关性。方法 将90只大鼠随机分为9组，每组10只，分别于去势后0、1、2、3、5、7、10、14、21d处死，取前列腺包埋切片后行免疫组织化学检测SGP－2和增殖细胞核抗原（PCNA),缺口末端标记术（TUNEL）和电镜检测前列腺细胞凋亡，计算阳性率，并作连续切片，行计算机图像重叠技术比较SGP－2表达与凋亡的相关性，及与PCNA表达的关系。结果 正常大鼠前列腺SGP－2表达较少，去势后则显著增加，与凋亡的变化及分布规律相似，其表达的阳性率与凋亡阳性率呈正相关（r＝0．8075，P＜0．01）。SGP－2的表达和PCNA呈负相关（r=－0．8061，P＜0．01）。结论 SGP－2在去势大鼠腹叶前列腺表达增高，与凋亡呈正相关，与PCNA呈负相关，提示可能有促凋亡和抑制增殖的作用。     

芹黄素对前列腺癌细胞生长和凋亡的影响

目的 :探讨芹黄素 (Apigenin)对前列腺癌 (PCa)细胞的体外杀伤效应 ,及其对前列腺癌 (PCa)细胞周期与凋亡的影响。方法 :以锥虫蓝拒染法进行细胞计数并计算芹黄素组与对照组细胞数比率 ,MTT法检测细胞生长抑制率 ,并采用流式细胞术 (FCM )检测细胞凋亡和细胞周期变化。结果 :芹黄素 0 .1μmol/L组细胞数未见明显改变 ,其余各组随浓度和时间的推移 ,细胞数比率逐渐下降 ,呈时间和剂量依赖关系 (P <0 .0 5～ 0 .0 1) ;这种作用在 1、10 μmol/L组可逆转 ,而在 10 0 μmol/L组不可逆转。MTT检测 1、10、10 0 μmol/L三个浓度组作用至2 4h后 ,细胞生长抑制率分别为 (5 .15± 0 .2 1) %、(10 .6 9± 1.80 ) %和 (18.16± 2 .34) % ;作用至 96h后 ,生长抑制率递增至 (2 4 .0 4± 3.34) % ,(36 .89± 2 .74 ) %和 (5 4 .18± 2 .6 0 ) %。芹黄素可使PC 3m细胞发生G0 /G1期细胞周期阻滞 ,高浓度组尤为明显 ,并在 10 0 μmol/L组诱导一定比例 (16 .19% )的细胞凋亡。结论 :芹黄素可能通过阻滞细胞周期和诱导细胞凋亡实现对PCa细胞的生长抑制作用。这为深入研究饮食结构与PCa的预防和治疗提供初步的实验依据。     

去势前后大鼠腹叶前列腺形态学的动态改变

目的：探讨去势前后大鼠腹叶前列腺形态学和组织学的变化规律，以及引起这种变化的机制。方法：将90只SD系成年雄性大鼠分为9组，其中一组作为正常对照，其余8组去势后分别于术后第1、2、3、5、7、10、14、21天处死，测血清雄激素浓度（T），腹叶前列腺的重量、体积和密度。再行石蜡包埋切片，HE染色，采用计算机图像分析系统计算腺体面积与间质面积之比值（G）、管腔分泌物与管腔面积这比值（S）、腺细胞高度（H）及单个腺体模截面细胞数（N）。TUNEL检测细胞凋亡，免疫组织化学方法测定增殖抗原PCNA。结果：去势后T急剧下降，前列腺重量和体积进行性减小，而密度先稍降低再逐渐升高。G、S、H、N均减小。腺上皮细胞凋亡、坏死，增殖下降。结论：去势后大鼠腹叶前列腺重量和体积的减小，可能是由于细胞数目减少、细胞萎缩、细胞分泌物减少等综合作用的结果，其始动因素可能是T水平的降低。     

Decrement of blood flow precedes the involution of the ventral prostate in the rat after castration

Blood flow to the rat ventral prostate (VP), dorsolateral prostate (DP), and Dunning R3327 prostatic tumors was measured at different times up to 7 days after castration, using the microsphere method. In the VP organ weight was decreased from day 3 onwards. Blood flow was, however, already significantly decreased from day 1. The reduced blood flow in VP in 1–3 and 7-day castrated animals could be reversed by testosterone treatment. Organ weight was slightly decreased but blood flow was unaffected by castration in DP. Castration left Dunning tumor volume and blood flow unaffected. Using immunohistochemistry, androgen receptors were observed in epithelial and stromal cells in VP, DP and Dunning tumors, but not in blood vessels. Castration is known to induce apoptosis in the VP, but not in the DP or in Dunning tumors. This suggests that a reduction in blood flow might be an important component for the castration-induced involution and apoptosis in prostatic tissue. The reason why castration reduces blood flow only in the VP, and not in the DP or Dunning tumor is unknown.     

Transient ischemia induces apoptosis in the ventral prostate of the rat

The mechanisms involved in the castration-induced involution of the ventral prostate (VP) are not fully understood. It was recently reported that castration decreases blood flow in the VP in rats and that this occurs before the apoptotic involution of the organ. However, it is unknown whether a decrease in blood flow may trigger apoptosis in the VP, and this was therefore examined in this study. The right iliac artery was clamped for 1 h in adult male rats. After 24 h of reperfusion, the VPs were frozen or fixed. In situ end-labeling (ISEL) was used to identify apoptotic cells, and testosterone repressed prostatic message-2 (TRPM-2) was measured. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was used to identify proliferating cells. Clamping the right iliac artery reduced blood flow in the right VP to 0.17 of that in the contralateral lobe. This relative ischemia resulted in a threefold increase in the volume density of apoptotic epithelial cells on the treated side, but left cell proliferation unaffected. Testosterone substitution did not change this pattern. This study suggests that a transient period of relative ischemia may induce apoptosis in the rat ventral prostate. This may be of importance for the understanding of castration-induced prostatic involution. Received: 26 June 1998 / Accepted 7 December 1998     

自噬在吉西他滨诱导胰腺癌细胞凋亡中的作用

目的:研究自噬在吉西他滨(gemcitabine,Gem)诱导胰腺癌细胞SW1990凋亡中的作用,并以氯喹特异性抑制自噬,以探讨其可能机制。方法:采用CCK8法检测Gem对胰腺癌细胞增殖的影响,并用实时定量PCR检测自噬相关基因LC3的表达,以p62免疫荧光染色检测细胞内自噬泡,通过Western印迹法检测自噬相关蛋白LC3、Beclin 1的表达,并用AnnexinⅤ/PI流式细胞方法检测Gem诱导后细胞凋亡的变化。进一步通过氯喹抑制自噬,检测自噬抑制前后细胞增殖、自噬及凋亡的变化。结果:Gem对胰腺癌细胞SW1990增殖具有部分抑制作用,Gem作用能迅速激活细胞自噬从而产生耐药。LC3的mRNA表达升高1.4~2.2倍(P<0.05),LC3-Ⅱ蛋白水平升高1.5~2.7倍(P<0.05)。Gem和氯喹联合用药组较Gem单药组细胞凋亡蛋白Caspase-3表达升高,细胞存活率从64.3%±3.1%降低为35.2%±3.4%(P<0.05)。结论:自噬在Gem诱导胰腺癌细胞凋亡的过程中可能起到保护作用,氯喹抑制自噬后可增强Gem的促凋亡作用。     

当归对大鼠椎间盘髓核细胞自噬及氧化应激损伤的作用及机制研究

目的 :探讨当归降低椎间盘髓核细胞自噬及氧化应激损伤的作用及其机制。方法 :从大鼠椎间盘中分离并培养SD大鼠原代髓核细胞,用不同浓度(0.1mg/ml、1mg/ml、5mg/ml)的当归注射液干预第3代细胞,噻唑蓝(methyl thiazolete trazolium,MTT)法检测适当的当归浓度。将原代髓核细胞随机分成3组:A组(对照组)、B组(H2O2组)和C组(当归+H2O2)组。A组用生理盐水处理24h,不进行H2O2刺激,B组用400μmol/L H2O2分别刺激0.5h、1.5h和3h,C组用适当浓度当归注射液预处理24h后,400μmol/L H2O2分别刺激0.5h、1.5h和3h。流式细胞术检测各组细胞不同时间的凋亡率,western blot检测凋亡相关蛋白细胞色素C、Bcl-2和Bax及自噬相关蛋白LC3-Ⅱ/Ⅰ、p62、p-AKT、p-m TOR和p-p70S6K的表达。结果:与其他浓度当归处理组比较,1mg/ml当归注射液可显著增加细胞存活(P0.05)。与A组相比,B组细胞凋亡率随着时间的延长逐渐增加,细胞色素C的表达增加(P0.05),Bcl-2/Bax的比例减小(P0.05),LC3-Ⅱ/Ⅰ的比例增加(P0.05),p62、p-AKT、p-m TOR和p-p70S6K的表达降低(P0.05);与B组相比,C组中细胞凋亡率和细胞色素C、p62、p-AKT、p-m TOR和pp70S6K的表达均降低(P0.05),Bcl-2/Bax和LC3-Ⅱ/Ⅰ的比例升高(P0.05)。结论 :1mg/ml当归注射液能够抑制大鼠髓核细胞凋亡,这可能与线粒体凋亡通路被阻断相关;1mg/ml当归能促进其髓核细胞自噬,这可能与AKT/m TOR信号通路被抑制相关。     

番茄红素促进前列腺癌LnCaP细胞早期凋亡

目的 观察类胡萝卜素中番茄红素对前列腺癌LnCaP细胞早期凋亡的影响。方法 采用磷脂结合蛋白荧光素标记法,结合流式细胞仪检测技术,分别测定不同浓度(0.5、5.0、10.0、15.0 μmol/L)番茄红素对前列腺癌LnCap细胞早期凋亡的影响,测量细胞早期凋亡的数目,比较不同浓度番茄红素与溶剂对照组早期凋亡细胞的差异。结果 加入不同浓度番茄红素的细胞组与溶剂对照组比较,LnCap细胞早期凋亡的比例分别是番茄红素组2.08％、1.10％、32.20％、9.61％,溶剂对照组0.65％、0.48％、13.10％、6.75％,可见加入番茄红素可使LnCap细胞早期凋亡率增加。结论 番茄红素有促进前列腺癌LnCaP细胞早期凋亡的作用。     

雷帕霉素调节自噬抑制凋亡促进骨折愈合的机制研究

目的探讨雷帕霉素调节细胞自噬抑制凋亡促进骨折愈合的机制。 方法建立SD大鼠右侧股骨干骨折模型,随机分为两组：空白对照组和雷帕霉素组（1 mg/kg/d）。空白对照组无特殊处理,雷帕霉素组在术后12 h、24 h,3 d和7 d腹腔注射1 mg/kg的雷帕霉素。分别在造模后2 w,4 w和6 w应用X线和Micro CT评估骨组织愈合情况,免疫荧光检测LC3-Ⅱ,免疫组化检测Beclin-1的表达水平,western blot检测Bax、VEGF及p-mTOR,HE染色计数骨折后骨组织中成骨细胞数量来评估成骨活性。 结果与对照组相比,雷帕霉素组骨折端骨组织中p-mTOR及p-mTOR/mTOR比值降低[2 w（t=6.703,P=0.004;t=16.346,P=0.000）、4 w（t=16.407,P=0.000;t=16.738,P=0.000）、6 w（t=6.101,P=0.001;t=6.104,P=0.001）];自噬蛋白LC3-Ⅱ和Beclin-1表达明显增加[2 w（t=9.509,P=0.000;t=9.962,P=0.000）、4 w（t=3.868,P=0.008;t=24.490,P=0.000）、6 w（t=5.071,P=0.002;t=12.454,P=0.001）];抗凋亡蛋白Bcl-2表达增加[2 w（t=-9.099,P=0.000）、4 w（t=-8.128,P=0.000）、6 w（t=-9.497,P=0.000）];促凋亡蛋白Bax表达降低[2 w（t=-4.263,P=0.005）、4 w（t=-2.936,P=0.026）和6 w（t=-8.343,P=0.000）];VEGF表达增加[2 w（t=-5.754,P=0.001）、4 w（t=-5.077,P=0.002）和6 w（t=-12.200,P=0.000）],差异均具有统计学意义。X线结果显示雷帕霉素组术后2 w、4 w和6 w Garrett评分高于对照组（t=4.371,P=0.005;t=5.166,P=0.002;t=4.243,P=0.005）,micro-CT结果显示雷帕霉素组术后2 w、4 w和6 w骨密度评分高于对照组（t=8.765,P=0.000;t=25.649,P=0.000;t=11.199,P=0.000）,差异具有统计学意义。 结论雷帕霉素通过诱导细胞自噬,抑制细胞凋亡促进骨折愈合。     

前列腺癌细胞凋亡与bcl-2蛋白表达及其意义

目的：探讨细胞凋亡及ｂｃｌ－２蛋白表达在前列腺癌（ＰＣａ）中的意义及前二者之间的相互关系。方法：采用原位细胞凋亡标记及免疫组织化学法对２５例ＰＣａ、１８例前列腺增生症 （ＢＰＨ）及１０例正常列腺的前列腺组织石蜡切片行ｂｃｌ－２蛋白及细胞凋亡检测。结果：ＰＣａ组的细胞凋亡充显著高于ＢＰＨ组及正常组,且ＰＣａ的细胞凋亡率与其分化程度呈显著负相关。ｂｃｌ－２蛋白在ＰＣａ组中表达率为３２％,与分化程度无关