功能矫形前伸青春期大鼠下颌后浅层嚼肌细胞凋亡的研究

目的:研究功能矫形前伸大鼠下颌后浅层嚼肌细胞凋亡的变化规律,探讨功能矫形的肌肉改建机理。方法:选用50只5周龄Sprague-Dawley(SD)雄性大白鼠,随机分为实验组和对照组各25只。实验组大鼠戴自制上颌功能矫治嚣,引导下颌前伸,并打开咬合。利用RT-PCR方法检测两组大鼠浅层嚼肌Bcl-2和Bax基因表达情况,利用TUNEL方法检测浅层嚼肌细胞凋亡情况。结果:①Bcl-2和Bax基因表达随大鼠戴用矫治器时间的延长而升高,至第3周开始下降但仍高于对照组,但Bax的表达高于Bcl-2。Bax/Bcl-2比值随大鼠戴用矫治器时间的延长而升高,至第4周开始下降。②TUNEL实验结果显示浅层嚼肌细胞在戴用矫治器1天后,开始出现凋亡,随着时间延长而增加,至第3周达到顶峰,第4周开始下降。结论:①Bax/Bcl-2比值升高促进浅层嚼肌细胞凋亡。②功能矫形可引起浅层嚼肌细胞凋亡,导致肌肉的结构和功能发生适应性改建。     

DDFA对重症急性胰腺炎大鼠肝脏细胞凋亡及Bax、Bcl-2基因表达的影响

目的探讨DDFA对重症急性胰腺炎(SAP)大鼠肝脏细胞凋亡及Bax、Bcl-2基因表达的影响。方法57只大鼠随机分为假手术组(SO组,n=9)、SAP组(n=24)和DDFA治疗组(n=24)。大鼠SAP模型采用50g/L牛磺胆酸钠胰腺被膜注射法建立,建模后6、12、24h测定血BIL、AST、ALT,光镜观察肝脏组织病理变化,TUNEL法测定肝脏细胞凋亡,SABC免疫组化染色法测定肝脏Bax、Bcl-2基因表达。结果SAP组BIL、AST、ALT和肝脏细胞凋亡指数、Bax基因表达较假手术组升高,Bcl-2基因表达较假手术组下降,光镜下见肝脏组织损害明显;经DDFA治疗后,BIL、AST、ALT和肝脏细胞凋亡指数、Bax基因表达下降,而Bcl-2基因表达增强,光镜下见肝脏组织损害减轻。结论肝脏细胞凋亡、Bax、Bcl-2基因表达参与SAP发病机制,在SAP早期给予DDFA治疗对减轻肝脏损害程度、改善预后是有益的。     

卡托普利对心力衰竭大鼠心肌细胞Bax、Bcl-2蛋白表达的影响

目的:探讨卡托普利对慢性压力负荷性心力衰竭大鼠心肌细胞凋亡相关基因Bax、Bcl-2蛋白表达的影响。方法:90只SD大鼠随机分为3组(n=30):假手术组(SH)、腹主动脉缩窄组(CAA)、卡托普利治疗组(CAP)。采用腹主动脉缩窄法复制模型,于第6、10周,检测各组心衰大鼠心肌细胞凋亡相关基因Bax,Bcl-2蛋白的表达。结果:造模后6周、10周结果均显示,CAA组较SH组心肌细胞凋亡基因Bcl-2蛋白及Bcl-2/Bax比例表达显著下降(P<0.01),Bax蛋白表达显著升高(P<0.01)。CAP组较CAA组Bcl-2蛋白及Bcl-2/Bax比例表达显著升高(P<0.01),Bax蛋白表达显著降低(P<0.01)。CAP组10周时较6周Bcl-2蛋白表达明显升高(P<0.05),Bax蛋白表达显著降低(P<0.01),Bcl-2/Bax比例显著升高(P<0.01)。结论:卡托普利能增加Bcl-2、降低Bax蛋白的表达,上调Bcl-2/Bax比率,从而抑制心肌细胞凋亡改善心功能。     

吲哚美辛对COPD 大鼠TNF-α 与Bcl-2 和Bax 表达的影响

目的:探讨吲哚美辛对慢性阻塞性肺疾病(COPD)模型大鼠趾长伸肌TNF-α及Bcl-2和Bax表达的影响。方法:100只健康Wistar大鼠随机分为模型组和对照组(A组)。模型组采用熏香烟和气管内滴注猪胰蛋白酶(PEE)法建立COPD模型大鼠,以低于A组大鼠平均体重的90%判断发生营养不良的标准,将模型组大鼠分为COPD营养正常组(B组)和COPD营养不良组,COPD营养不良组随机化原则分为COPD营养不良生理盐水组(C组)、吲哚美辛1组(D组)、吲哚美辛2组(E组)、吲哚美辛3组(F组)。A、B、C三组灌注等量生理盐水,D、E、F组给予不同剂量吲哚美辛(IND)灌胃干预。TUNEL法测定趾长伸肌细胞凋亡率,免疫组织化学法测定Bcl-2、Bax蛋白表达,双抗体夹心法测定血清及趾长伸肌肿瘤坏死因子α(TNF-α)浓度。结果:吲哚美辛干预后,E组趾长伸肌细胞凋亡率、Bcl-2、Bax的蛋白表达、趾长伸肌匀浆和吲哚美辛干预后血清TNF-α浓度低于C、D、F组,但高于A、B组,P<0.05;E组趾长伸肌Bcl-2蛋白表达高于C、D、F组,低于A、B组,P<0.05。结论:Bcl-2、Bax及TNF-α参与COPD模型大鼠趾长伸肌细胞凋亡,适当剂量的吲哚美辛可改善COPD营养不良模型骨大鼠趾长伸肌的凋亡。     

PTSD与杏仁核神经元细胞凋亡的关系

目的观察创伤后应激障碍（PTSD）大鼠杏仁核神经元细胞凋亡相关基因的表达与细胞凋亡的发生,从杏仁核神经元细胞凋亡揭示PTSD的部分发病机制。方法成年健康雄性Wister大鼠50只,随机分为连续单一刺激（single prolonged stress,SPS）模型的1d、4d、7d、14d组及正常对照组。应用免疫组化和Western Blotting技术检测凋亡相关基因Bax、Bcl-2在PTSD杏仁核神经元的表达;采用TUNEL法检测PTSD大鼠杏仁核神经元细胞凋亡。结果PTSD大鼠杏仁核神经元凋亡相关基因Bax于4d达高峰,Bcl-2于1d表达最高,Bax／Bcl-2比值逐渐升高,于4d达到峰值,之后渐趋下降。TUNEL阳性细胞在SPS各组模型均出现,4d达最高峰。结论PTSD大鼠杏仁核神经元细胞凋亡中,凋亡相关基因Bax、Bcl-2各自发挥促进凋亡和抑制凋亡的作用,Bax／Bcl-2比值升高促进细胞发生凋亡,可能与杏仁核调节的PTSD恐惧异常的发病机制相关。     

内毒素血症时大鼠心肌细胞凋亡与血管紧张素受体表达关系的研究

目的:对脂多糖诱导大鼠内毒素血症时心肌细胞凋亡与血管紧张素II受体AT1、AT2表达变化进行分析。方法:Wistar大鼠随机分为3组(n=10):空白组、脂多糖(LPS)干预2 h(L2)组、LPS干预6 h(L6)组。根据分组情况经腹腔注射LPS(10 mg/kg)制备内毒素血症模型,对照组给予等体积生理盐水腹腔注射。应用免疫组织化学法检测凋亡相关因子Bcl-2和Bax蛋白表达,并通过TUNEL法检测心肌细胞凋亡变化,并采用免疫组织化学、RT-PCR方法观察了大鼠心肌AT1、AT2受体表达变化,电镜观察心肌组织病理变化。结果:LPS干预2 h后Bcl-2阳性细胞表达开始明显上调,之后开始下降,但仍高于对照组(P<0.05)。Bax的表达随着干预时间的延长不断增加,6 h后表达与对照组相比,有显著的统计学差异(P<0.01)。Bcl-2/Bax的比率在干预开始2 h时升高,随后开始下降,心肌细胞凋亡增加。随着LPS干预的时间延长,AT1受体、AT2受体蛋白、mRNA表达水平呈下降趋势,6 h后表达与对照组相比,有显著的统计学差异(P<0.01)。结论:大鼠经LPS干预后随时间延长Bcl-2/Bax的比率呈下降趋势,心肌细胞凋亡增加,与AT1受体、AT2受体表达水平下降呈负相关,说明血管紧张素受体表达下调可能与内毒素血症时大鼠心脏功能损伤有关,为临床预防及治疗全身炎症反应所致心功能损伤提供一条新的思路以及实验室理论基础。     

IL-10 基因对糖尿病大鼠胰岛beta细胞凋亡的保护作用

目的：研究白细胞介素10（IL-10）基因对链脲佐菌素(STZ)诱导的糖尿病大鼠胰腺炎症浸润程度及胰腺组织中Bcl-2 及Bax 表达的影响。方法：建立链脲佐菌素性糖尿病模型,腺病毒介导的IL-10 基因(Ad-mIL-10)腹腔注射。检测大鼠空腹血糖值;免疫组 织化学法观察胰腺炎症浸润程度;TUNEL法检测胰岛细胞凋亡;免疫组化方法观察Ad-mIL-10 对实验性糖尿病大鼠胰岛凋亡调 控基因Bax 和Bcl-2 表达的影响。结果：Ad-mIL-10 腹腔注射糖尿病发病率低,平均血糖水平低,可以降低胰腺炎症浸润程度,减 少胰岛细胞凋亡。给予Ad-mIL-10 后大鼠Bax 基因的表达明显下降, Bcl-2 与Bax 的比值明显增加。结论：IL-10基因对实验性糖 尿病大鼠有降血糖作用,减少胰岛细胞凋亡,与调节Bcl-2 与Bax 基因的表达有关。     

IL-10基因对糖尿病大鼠胰岛β细胞凋亡的保护作用

目的：研究白细胞介素10（IL-10）基因对链脲佐菌素（STZ）诱导的糖尿病大鼠胰腺炎症浸润程度及胰腺组织中Bcl-2及Bax表达的影响。方法：建立链脲佐茵素性糖尿病模型,腺病毒介导的IL-10基因（Ad-mIL-10）腹腔注射。检测大鼠空腹血糖值;免疫组织化学法观察胰腺炎症浸润程度;TUNEL法检测胰岛细胞凋亡;免疫组化方法观察Ad-mIL-10对实验性糖尿病大鼠胰岛凋亡调控基因Bax和Bcl．2表达的影响。结果：Ad-mlL-10腹腔注射糖尿病发病率低,平均血糖水平低,可以降低胰腺炎症浸润程度,减少胰岛细胞凋亡。给予Ad-mlL-10后大鼠Bax基因的表达明显下降,Bcl-2与Bax的比值明显增加。结论：IL-10基因对实验性糖尿病大鼠有降血糖作用,减少胰岛细胞凋亡,与调节Bcl-2与Bax基因的表达有关。     

神经节苷脂GM1对帕金森模型小鼠黑质细胞凋亡及Bcl-2和Bax表达的影响

目的观察神经节苷脂GM1对帕金森模型小鼠黑质细胞凋亡及Bcl-2和Bax表达的影响,探讨其预防帕金森病的机制。方法成年C57-BL雄性小鼠分为正常对照组、模型组和神经节苷脂GMI治疗组。应用TUNEL法和免疫荧光组织化学染色技术观察各组小鼠黑质神经元凋亡及Bcl-2和Bax表达情况。结果TUNEL、Bcl-2和Bax阳性神经元主要位于黑质致密部。模型组黑质神经元凋亡数明显多于正常对照组,神经节苷脂GM1组黑质神经元凋亡数明显少于模型组。在模型组Bcl-2阳性神经元和Bax阳性神经元的数量均较正常对照组明显增多;在神经节苷脂GM1组Bcl-2阳性神经元的数量较模型组明显增多,但Bax阳性神经元的数量较模型组明显减少。结论神经节苷脂GM1可能通过增强黑质神经元内Bcl-2基因表达及抑制Bax基因表达的途径抑制黑质神经元的凋亡。     

维生素E对老年大鼠卵巢颗粒细胞保护作用机制的探讨

目的：通过观察维生素E（VE）对老年雌性大鼠卵巢凋亡相关蛋白Bcl-2、Bax的影响及对其抗氧化能力的影响,探讨VE延缓卵巢衰老的作用和机制。方法：采用自然衰老雌性大鼠,给予不同剂量外源性VE,用免疫组化方法观察卵巢颗粒细胞凋亡调控蛋白Bcl-2、Bax的表达,用Western Blot法检测卵巢Bcl-2、Bax蛋白含量,用生化法测定血清总超氧化物歧化酶（SOD）活力和丙二醛（MDA）含量。结果：与成年对照组相比,老年对照组Bcl-2表达明显降低、Bax表达明显升高（P〈0．01）,血清中SOD活力下降、MDA含量显著升高（P〈0．01）。与老年对照组相比,VE纽Bcl-2表达升高,Bax表达降低（P〈0．05）,MDA含量显著减少（P〈0．01）。结论：VE可调控凋亡相关蛋白Bcl-2、Bax的表达和对抗自由基对颗粒细胞的损伤,对卵巢颗粒细胞具有一定的保护作用。     

软脂酸对胰岛β细胞凋亡及Akt途径的影响

目的：探讨软脂酸（PA）对胰岛β细胞（MIN6细胞）凋亡及Akt信号途径的影响。方法：细胞采用小鼠胰岛素瘤细胞株MIN6,不同浓度PA（0-1．6mmol／L）干预24、48h,MTT、法测定各组细胞存活率。干预48h后Hoechst-PI染色法和Annexin—V／PI双标流式测定法测定各组细胞凋亡率,Western-blot法测定p-Akt、Akt、Bax、Bcl-2。结果：随着PA浓度的增高①MIN6细胞存活率逐渐减小、凋亡率逐渐增大;②MIN6细胞中p-Akt和Bcl-2的表达逐渐减少,而Akt、Bax无明显改变。结论：长时间PA作用引起MIN6细胞凋亡,并呈现一定的量效关系;这一效应可能是通过Akt／Bcl-2产生作用的。     

Expressions and localizations of Bax/Bcl-2 proteins during metamorphosis of Pelophylax ridibundus

Bcl-2 and Bax proteins are expressed in cells of the tails of Pelophylax ridibundus larvae. We investigated the levels of these proteins in tails undergoing apoptosis. Apoptotic cells were observed in the epidermis, muscle and notochord of tails of different lengths. The apoptotic cells in epidermis exhibited the typical features of apoptosis. Amorphous masses and irregularities in striated muscle tissue undergoing apoptosis and apoptotic remnants in the notochord also were observed. In general, Bax staining in the epidermis, subepidermal fibroblast layer, muscle and notochord cells increased, while Bcl-2 staining decreased as the tail regressed. Our results suggest that during tail regression due to metamorphosis, Bcl-2 and Bax proteins play key roles in the apoptosis of tail epidermis, subepidermal fibroblast layer, muscle and notochord cells.     

木犀草素对子痫前期大鼠滋养层细胞凋亡的影响及其机制

目的探讨木犀草素（LUT）对子痫前期（PE）大鼠滋养层细胞凋亡的影响及其机制。 方法取妊娠10 d SD大鼠,按随机数字表法随机分为对照组、模型组、20、40、60 mmol/L LUT （LUT-L、LUT-M、LUT-H）组,每组各12只,模型组和给药组大鼠皮下注射100 mg/（kg·d）亚硝基左旋精氨酸甲酯建立PE大鼠模型,对照组大鼠皮下注射等量生理盐水,每天1次,注射6 d。妊娠16 d的大鼠分别予以20、40、60 mmol/L LUT腹腔注射,对照组、模型组大鼠腹腔注射等量生理盐水,每天1次,注射5 d。测量各组大鼠妊娠10、16、21 d尾动脉血压及24 h尿蛋白水平;妊娠21 d,原位末端标记法（TUNEL）检测滋养层组织细胞凋亡情况,Western blot法检测滋养层组织B淋巴细胞瘤-2 （Bcl-2）、Bcl-2相关X蛋（Bax）、磷脂酰肌醇3-激酶（PI3K）、磷酸化PI3K （p-PI3K）、蛋白激酶B （Akt）、磷酸化AKT （p-Akt）、内皮型一氧化氮合酶（eNOS）和磷酸化eNOS （p-eNOS）蛋白表达量。多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。 结果妊娠10 d,各组大鼠尾动脉收缩压、舒张压、24 h尿蛋白含量差异无统计学意义;妊娠16 d,与对照组比较,模型组、LUT-L组、LUT-M组、LUT-H组大鼠尾动脉收缩压、舒张压、24 h尿蛋白含量升高（P均< 0.05）;妊娠21 d,与对照组比较,模型组、LUT-L组、LUT-M组、LUT-H组收缩压[（110.33±3.67）比（147.28±4.16）,（131.29±4.31）,（124.46±4.27）,（118.54±4.18）mmHg]、24 h蛋白尿、细胞凋亡率[（1.38±0.34）%,（43.45±3.72）%,（39.21±3.53）%,（27.86±3.41）%,（23.21±3.28）%]和Bax蛋白表达量均升高;Bcl-2、p-PI3K/PI3K （1.06±0.09比0.25±0.02,0.37±0.03,0.57±0.06,0.73±0.08）、p-Akt/Akt（0.87±0.08比0.11±0.01,0.23±0.03,0.56±0.07,0.78±0.06）和p-eNOS/eNOS蛋白表达水平（0.85±0.07比0.09±0.01,0.16±0.02,0.38±0.04,0.69±0.07）均降低（P均< 0.05）。与模型组比较,LUT-L组、LUT-M组、LUT-H组大鼠尾动脉收缩压、舒张压、滋养层组织细胞凋亡率和Bax蛋白表达量降低,Bcl-2、p-PI3K/PI3K、p-Akt/Akt和p-eNOS/eNOS蛋白表达量升高（P均< 0.05）。 结论LUT可抑制PE大鼠滋养层组织细胞凋亡,其机制可能与PI3K/Akt/eNOS信号通路激活,调控凋亡相关蛋白表达有关。     

小鼠生精细胞增殖与凋亡的年龄变化

为研究雄性小鼠睾丸在发生,发育过程生精细胞增殖与凋亡的年龄变化,本研究采用了免疫细胞化学,凋亡细胞原位检测,电镜及体视学图象分析等方法,对胚胎15天到生后10月后发育阶段生精细胞的超微结构,PCNA表达,凋亡情况进行了较深入地研究,结果：（1）精原细胞PCNA反应在胚胎15天为阳性,从胚胎18天到生后5天,降为弱阳性,而阳性的精原细胞在生后7天重新出现,一直到生后6月,仍可见部分精原细胞呈阳性反应;（2）生后3天,可见凋亡的精原细胞染色质浓缩,核膜出现明显的核周隙,核碎裂,凋亡细胞数从生后1天到生后第3周有增加的趋势,于生后第3周出现峰值,之后降低,之后降低,结论：（1）PCNA阳性细胞而密度到生后第2周出现峰值,而凋亡细胞数于生后3周出现峰值,生精细胞凋亡的高峰要滞后其增殖峰1周左右,而且与其所处生精周期的特定阶段有关;（2）精原细胞在胚胎发育过程中向曲细精管周边迁移,其排列由无序到有序;（3）在生后各发育 ,精原细胞始终保持DNA复制的能力。     

The timing of re-institution of good blood glucose control affects apoptosis and expression of Bax and Bcl-2 in the retina of diabetic rats

Hyperglycemia initiates a sequence of events that leads to the development of diabetic retinopathy. We explored the effect of re-institution of good blood glucose control on apoptosis and apoptosis related genes (Bax and Bcl-2) in the retina of diabetic rats. Fifty male Wistar rats randomly divided into five groups : normal control group (CON), diabetic rats with high blood glucose levels for 8 months group (DM) ,diabetic rats with good blood glucose control for 8 months group (DM₁),diabetic rats with poor blood glucose control for 2 month followed by good blood glucose control for six additional months group (DM₂), rats with poor blood glucose control for 4 months followed by good blood glucose levels for four additional months group (DM₃). Expression of Bax and Bcl-2 in the retina was studied by immunohistochemistry and the apoptotic cells were stained using the TUNEL method. The apoptotic cell, expression of Bax and Bcl-2 and the ratio of Bax to Bcl-2 in the retina was increased in DM group compared with normal rats’ (P < 0.01). There was no significant difference in apoptotic cells and the ratio of Bax to Bcl-2 between DM₁ group and CON group. The number of TUNEL positive cells and Bax to Bcl-2 ratio was partially reversed in DM₂ group. But glucose control had no effect on the apoptotic cells and the expression of Bax and Bcl-2 in DM₃ group. There was a positive correlation between apoptotic cells and Bax/Bcl-2 ratio in the retina (r = 0.808, P < 0.01). Good blood glucose control at early stage can decrease the number of apoptotic cells in the retina; the decreased apoptosis is correlated with the down-regulation of Bax to Bcl-2 ratio.     

Apoptotic responses to hindlimb suspension in gastrocnemius muscles from young adult and aged rats

Although apoptosis has been demonstrated in soleus during hindlimb suspension (HS), it is not known whether apoptosis is also involved in the loss of muscles dominated by mixed fibers. Therefore, we examined the apoptotic responses in gastrocnemius muscles of young adult and aged Fischer 344 x Brown Norway rats after 14 days of HS. The medial gastrocnemius muscle wet weight significantly decreased by 30 and 32%, and muscle wet weight normalized to the animal body weight decreased by 11 and 15% in young adult and aged animals, respectively, after HS. The extent of apoptotic DNA fragmentation increased by 119 and 61% in suspended muscles from young and aged rats, respectively. Bax mRNA increased by 73% in young muscles after HS. Bax and Bcl-2 protein levels were greater in suspended muscles relative to control muscles in both age groups. The level of cytosolic mitochondria-housed apoptotic factor cytochrome c was significantly increased in the mitochondria-free cytosol of suspended muscles from young and aged rats. In contrast, the release/accumulation of AIF, a caspase-independent apoptogenic factor, was exclusively expressed in the suspended muscles from aged rats. Our data also show that aging favors the proapoptotic signaling in skeletal muscle by altering the contents of Bax, Bcl-2, Apaf-1, AIF, caspases, XIAP, Smac/DIABLO, and cytochrome c. Furthermore, these results indicate that apoptosis occurs not only in slow-twitch soleus muscle but also in the mixed-fiber (predominately fast fibered) gastrocnemius muscle. Our data are consistent with the hypothesis that apoptotic signaling differs in young adult and aged gastrocnemius muscles during HS.     

缺血后处理对大鼠再灌注损伤肺细胞凋亡的影响

目的：探讨缺血后处理对再灌注损伤肺细胞凋亡的影响。方法：健康雄性sD大鼠24只,随机分为对照组（C组）、缺血／再灌注组（I／n组）和缺血后处理组（IPostC组）（n=8）。对比观察各组血清中丙二醛（MDA）、超氧化物歧化酶（SOD）、髓过氧化物酶（MPO）活力及含量变化,原位缺口末端标记法（TUNEL）检测肺组织细胞凋亡情况,免疫组化及RT-PCR法检测肺组织中Bax、Bcl一2蛋白和基因的表达。结果：I／R组与c组相比,MDA含量、MPO活力明显升高,SOD活力明显下降（均P〈0．01）,肺组织原位细胞凋亡检测示I／R组凋亡指数（AI）（39．03±3．46）显著高于C组（2．88±0．34）,Bcl-2／Bax比值在蛋白和基因水平明显降低（均P〈0．01）;IPostC组与I／R组相比MDA含量显著降低（P〈0．05）,MPO活力显著降低（P〈0．01）,SOD活性升高（P〈0．01）,AI为8．03±0．88显著低于L／R组,并能明显升高Bcl-2／Bax比值（均P〈0．01）。结论：缺血后处理通过减轻脂质过氧化反应及中性粒细胞聚集,降低Bax／Bel．2比值,使肺组织细胞凋亡减少,从而有效地减轻肺缺血／再灌注损伤。     

Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

Muscle cell apoptosis accompanies normal muscle development and regeneration, as well as degenerative diseases and aging. C2C12 murine myoblast cells represent a common model to study muscle differentiation. Though it was already shown that myogenic differentiation of C2C12 cells is accompanied by enhanced apoptosis in a fraction of cells, either the cell population sensitive to apoptosis or regulatory mechanisms for the apoptotic response are unclear so far. In the current study we characterize apoptotic phenotypes of different types of C2C12 cells at all stages of differentiation, and report here that myotubes of differentiated C2C12 cells with low levels of anti-apoptotic Bcl-2 expression are particularly vulnerable to apoptosis even though they are displaying low levels of pro-apoptotic proteins Bax, Bak and Bad. In contrast, reserve cells exhibit higher levels of Bcl-2 and high resistance to apoptosis. The transfection of proliferating myoblasts with Bcl-2 prior to differentiation did not protect against spontaneous apoptosis accompanying differentiation of C2C12 cells but led to Bcl-2 overexpression in myotubes and to significant protection from apoptotic cell loss caused by exposure to hydrogen peroxide. Overall, our data advocate for a Bcl-2-dependent mechanism of apoptosis in differentiated muscle cells. However, downstream processes for spontaneous and hydrogen peroxide induced apoptosis are not completely similar. Apoptosis in differentiating myoblasts and myotubes is regulated not through interaction of Bcl-2 with pro-apoptotic Bcl-2 family proteins such as Bax, Bak, and Bad.     

Altered Sensitivity to Retinoid-Induced Apoptosis Associated with Changes in the Subcellular Distribution of Bcl-2

In the acute promyelocytic leukemia cell line NB4, Bcl-2 downregulation occurred as a late event of retinoid-induced differentiation. In the maturation-resistant NB4-R1 subclone, retinoids failed to downregulate Bcl-2 even in the situation of apoptosis massively induced by pan-agonists and RXR-selective agonists. We observed that NB4 and NB4-R1 cells differed with respect to the intracellular localization of Bcl-2 which showed a perinuclear localization in NB4-R1 cells, while Bax was broadly expressed in the cytoplasm and to only a minor extent in the perinuclear area. Therefore, the distinct intracellular localization of Bcl-2 and Bax was in general nonoverlaping. Bcl-2 remained massively expressed until cell disruption. Bax was not significantly upregulated in cells committed to death. However, Bax localization changed from a diffuse pattern to concentrate in few specific cytoplasmic area at a stage preceding the formation of apoptotic bodies. A human Bcl-2 transgene was transiently overexpressed in NB4-R1 cells which showed increased resistance to apoptosis induced by retinoids. Stably transfected clones of NB4-R1 cells showed an increased expression of Bcl-2 and a marked resistance to apoptosis. Interestingly, the overexpression of Bcl-2 restored a pattern of uniform Bcl-2 labeling in the cytoplasm and, remarkably, the colocalization of Bcl-2 with Bax. This work demonstrates that the ability of retinoid-induced cells to undergo apoptosis depends on the level of expression and the functional interaction between Bcl-2 and Bax.