腹腔镜根治性膀胱前列腺切除术治疗前列腺癌广泛侵及膀胱6例报告北大核心CSCD

目的 探讨腹腔镜根治性膀胱前列腺切除术治疗前列腺癌广泛侵犯膀胱的疗效。方法 2015年12月~2019年6月我们对6例前列腺癌广泛侵及膀胱行腹腔镜下根治性膀胱前列腺切除及尿流改道手术,采用经腹腔入路五孔法,游离输尿管中下段至膀胱壁外,由髂总动脉分叉处开始,清扫两侧髂外动脉旁淋巴结、闭孔淋巴结和髂内动脉旁淋巴结,游离输精管、精囊及前列腺后面,游离膀胱前壁,缝扎阴茎背深静脉复合体,游离膀胱侧韧带及前列腺侧韧带,离断尿道,在腹腔镜下完成膀胱前列腺切除和淋巴结清扫,后距回盲处近端约20 cm处取长约15 cm回肠,体外构建流出道。术后均行辅助内分泌治疗。结果 6例腹腔镜下根治性膀胱前列腺切除联合回肠膀胱通道术顺利完成,无中转开放手术及输血。手术时间(322.5±41.2)min,术后住院时间(10.8±2.5)d,无Ⅲ级及以上并发症。术后切缘阳性4例,淋巴结阳性4例;术后病理分期T4N1M0期4例,T4N0M0期2例。术后局部症状改善明显,局部症状评分平均0.5分(0~1分),低于术前(平均3.2分)。术后1个月血肌酐(109.8±23.7)mmol/L。6例中位随访45(30~72)个月,1例死于心脏疾病,2例PSA进展,辅助放疗及多西他赛治疗,3例未见复发或转移。结论 腹腔镜根治性膀胱前列腺切除术可作为前列腺癌广泛累及膀胱的一种治疗选择,能改善患者局部症状。     

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

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.     

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.     

树突状细胞与膀胱癌关系研究进展

树突状细胞(Dendritic cell,DC)是专职的抗原呈递细胞(APC)，DC在机体抗肿瘤中起重要作用。在我国，膀胱癌是泌尿系最常见的肿瘤。本文就DC与肿瘤免疫逃避关系，采用DC治疗膀胱癌的临床应用状况及存在问题作一综述。     

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.     

F344大鼠树突状细胞的分离与培养

目的　研究F344大鼠树突状细胞(DC)的分离与培养方法。方法　取大鼠四肢骨骨髓细胞悬液,经梯度离心法得到DC ,加入白细胞介素(IL) - 4、粒细胞巨噬细胞集落刺激因子(GM CSF) ,全RPMI 16 4 0培养,经倒置显微镜、电镜及流式细胞仪鉴定细胞纯度及成熟度。结果　细胞培养的第8天,经倒置显微镜、电镜观察DC出现典型形态;流式细胞仪检测第4天OX6 2阳性率为8 .92 %、主要组织相容性复合物(MHC) Ⅱ为2 0 . 90 %、CD86为16 98% ;第8天OX6 2阳性率为5 8. 0 7%、MHC Ⅱ为6 0 . 4 9%、CD86为6 2 . 94 % ;第15天OX6 2阳性率为84 . 6 8%、MHC Ⅱ为88 .0 3%、CD86为6 2 . 80 %。结论　用该方法得到的DC纯度可达80 %以上,为进一步研究其在肿瘤研究中的应用打下基础。     

F344大鼠膀胱肿瘤模型的制备与检测

目的通过膀胱灌注N-甲基亚硝基脲(MNU)诱导F344大鼠膀胱肿瘤模型。方法选取F344大鼠40只,随机分为两组第1组膀胱灌注生理盐水(对照组),第2组膀胱灌注MNU(MNU组),每2周1次,共5次;于第14周处死动物,取血清测干扰素(INF-γ),膀胱肿瘤组织测主要组织相容性抗原-I(MHC-I)及病理观察。结果MNU组出现膀胱肿瘤,对照组无1例肿瘤;对照组血清INF-γ为(9.620±7.256)pg/ml,实验组为(0.898±0.323)pg/ml,两组相比有统计学意义(P<0.001)。实验组膀胱肿瘤的MHC-I表达较高(58.60%～93.80%)。结论MNU可成功诱导F344大鼠膀胱肿瘤,该模型是肿瘤免疫治疗的理想模型。