Detection of apoptotic cells and immunohistochemical study of bcl-2 and p53 gene protein in primary gastric mucosa-associated lymphoid tissue (MALT) lymphoma

To identify the apoptotic cells in gastric MALT lymphoma and its relationship between bcl-2 and p53 gene expression. Methods: TdT-mediated dUTP biotin Nick End labeling (TUNEL) and immuno-histochemistry ABC method were used to display apoptotic cells and the gene protein expression of bcl-2 and p53 independently. Results: Apoptotic indices (AI) in high-grade MALT lymphomas were significantly higher than in mixed-grade group and low-grade group (P<0.05). Bcl-2 was expressed in 83% of low-grade tumors, 61.6% of the median-grade tumors and 43.7% of high-grade tumors. An inverse correlation was observed between the expression of bcl-2 and apoptotic indices. Only 27 cases were p53 positive. The frequency of p53 positivity was significantly increased as the histologic grade advanced (P<0.05). There was also an inverse correlation between the expression of bcl-2 and p53. Conclusion: Apoptosis may be important in tumors development and transmission. p53 and bcl-2 were important regulatory genes of apoptosis and may be associated with transformation from low- grade to high-grade lymphomas.

抗凋亡蛋白Bcl-2在非霍奇金淋巴瘤中的表达及其临床意义

目的:通过抗凋亡蛋白Bcl-2的检测,观察其在非霍奇金淋巴瘤（NHL）中的表达及临床意义。方法:用免疫组织化学染色的方法,对34例NHL外检石蜡包埋组织切片进行Bcl-2蛋白的检测。结果:（1）低度恶性NHL中Bcl-2表达高于高度恶性组（P<0.005）;（2）B细胞性NHL中Bcl-2的表达高于T细胞性NHL（P<0.025）;（3）首次化疗缓解组中的Bcl-2表达低于未缓解组（P<0.01）;（4）高Bcl-2表达组的生存期与低表达组之间无差异。结论:抗凋亡蛋白Bcl-2的表达与NHL恶性程度及化疗缓解率相关,可能成为NHL预后的判断指标。

儿童非霍奇金淋巴瘤EB病毒LMP-1和P^(53)、bcl-2表达的关系

目的 探讨儿童NHL EB病毒LMP-1和P^(53)、bcl-2蛋白的表达及关系。方法 采用免疫组化Envision法检测64例儿童NHL中LMP-1和P^(53)、bcl-2蛋白。结果(1)P^(53)蛋白阳性表达39例(60.9％),表达强度与淋巴瘤恶性程度呈正相关;阳性表达率在低恶组与中、高恶性组间有显著性意义,P<0.01。bcl-2蛋白阳性表达37例(53.8％),bcl高于TCL,低恶性高于高恶性。(2)LMP-1蛋白阳性表达45例(70.3％),阳性表达率与肿瘤恶性程度和年龄有统计学意义,P<0.01;而与淋巴瘤免疫表型、性别和发病部位无关。LMP-1表达与P^(53)及bcl-2的表达呈正相关。结论 EBV感染是儿童NHL发生发展不可忽视的病毒致病因素,其致病作用可能是通过上调P^(53)、bcl-2蛋白实现的。

高脂血症大鼠脑缺血/再灌注后p38 MAPK活化及对Bax和Bcl-2表达的影响

目的检测高脂血症大鼠脑缺血/再灌注(I/R)损伤后大脑皮质内磷酸化p38 MAPK(p-p38 MAPK)、Bax和Bcl-2表达变化,及SB203580对p-p38 MAPK、Bax和Bcl-2表达的影响,研究在高脂血症脑I/R损伤中p38 MAPK活化对Bax和Bcl-2表达的影响。方法大鼠高脂血症模型建立后,将其随机分为3组,假手术组(sham)、手术组(I/R)、SB203580处理组(SB+I/R),每组10只。线栓法建立左侧大脑中动脉栓塞I/R模型,神经行为学评分观察大鼠神经行为损伤症状,2,3,5-氯化三苯基四氮唑(TTC)染色显示脑梗死灶,TUNEL染色观察凋亡细胞,免疫组织化学法分析p-p38 MAPK、Bax和Bcl-2相对表达水平。结果与sham组比较,I/R组大鼠脑梗死体积百分比、细胞凋亡指数和神经行为学评分均显著升高,且p-p38 MAPK、Bax表达明显增高,Bcl-2表达明显降低,差异均具有统计学意义(P<0.05)。与I/R组比较,SB+I/R组大鼠脑组织损伤减轻,梗死灶明显缩小,细胞凋亡指数明显降低,p-p38 MAPK表达明显降低,Bax表达减少而Bcl-2表达增多,差异均有统计学意义(P<0.05)。SB+I/R组较I/R组神经行为学评分降低,但差异无统计学意义。结论在高脂血症大鼠脑缺血再灌注损伤过程中,p38 MAPK活化可调节Bax和Bcl-2的表达。

Bcl-2蛋白在B细胞淋巴瘤中的表达

目的 研究B细胞淋巴瘤与bcl 2蛋白表达的关系。方法 采用免疫组织化学技术检测 6 3例B细胞淋巴瘤中bcl 2蛋白的表达。结果 6 3例B细胞淋巴瘤bcl 2蛋白 (+ ) 4 9例 (77.8% ) ,低、高恶性及结内、外淋巴瘤之间的bcl 2阳性率均无显著性差异 (P >0 .0 5 )。结论 bcl 2蛋白的表达与B细胞淋巴瘤关系较密切 ,但与恶性程度、病变部位及类型无明显关系。

大鼠自发性乳腺肿瘤中Bax、Bcl-2和Caspase-3的表达

通过130只(雌雄对半)Sprague-Dawley(SD)大鼠进行自发性乳腺肿瘤造模,并研究自发产生的乳腺肿瘤组织的病理变化及B细胞淋巴瘤/白血病-2(B cell lymphoma/leukmia-2,Bcl-2)、B细胞淋巴瘤/白血病-2相关X蛋白(B cell lymphoma/leukmia-2 associated X protein,Bax)和含半胱氨酸的天冬氨酸蛋白水解酶-3(cysteinyl aspartate specific proteinase-3,Caspase-3)在乳腺肿瘤中的蛋白和m RNA表达变化。造模结果显示共有14只雌性大鼠发生乳腺肿瘤,对总体而言肿瘤的发病率为10.77%(14/130);由于雄性大鼠未见乳腺肿瘤,在雌性大鼠中肿瘤的发病率为21.54%(14/65)。苏木精-伊红(hematoxylin-eosin,HE)染色病理切片观察确定肿瘤分型分别为乳腺纤维腺瘤7例,乳腺腺癌3例,乳腺乳头状癌2例,乳腺浸润性导管癌2例。免疫组织化学(immunohistochemistry,IHC)结果显示,对照组(n=5)Bax、Bcl-2和Caspase-3的蛋白表达量分别为9.75±0.04、8.13±0.35和6.60±0.04,而这3个因子在良性病变组(n=7)的蛋白表达量分别为8.16±0.16、9.14±0.22和4.43±0.1。另外,其在恶性病变组(n=7)的蛋白表达量分别为6.49±0.32、9.24±0.81和3.03±0.10,与对照组相比,Bax和Caspase-3在病变组的表达极显著减少(P<0.01),而Bcl-2在病变组中的表达与对照组相比也显著增加(P<0.05),且正常对照组Bcl-2/Bax比值小于1,而在良性组和恶性组则均大于1。荧光定量聚合酶链式反应(real-time fluorescence quantitative PCR,RTFQ-PCR)结果通过相对定量计算显示,Bax、Bcl-2和Caspase-3在各组中的m RNA的表达量变化与蛋白表达一致,相比差异有高度统计学意义(P<0.01)。综上所述,自发性乳腺肿瘤在雌性大鼠中的发病率较高,且病变类型多样,而雄性发病率为0;且Bcl-2、Bax和Caspase-3与大鼠自发性乳腺肿瘤的发生有着密切的联系,从而为临床乳腺肿瘤的诊断、治疗和预后提供了新的思考方向。

THE EXPRESSION AND CLINICAL VALUE OF APOPTOSIS CONTROL GENE Bcl-2 AND Bax IN BREAST CANCER

Objective: To study the expression and clinical value of apoptosis control gene bcl-2 and bax in breast cancer. Methods: Protein bax and bcl-2 in 41 breast cancers obtained from operations in our hospital in 1996 were detected using ABC immunohistochemical stain assay and compared with 10 cases with normal breast tissues. Results: The positive rate of bax in normal breast tissue was 90% and in breast cancer was 59%, with a significant statistical difference between them (P<0.05), but there was no statistical difference in bcl-2 protein expression. Among the 41 breast cancer, the group with lymph node metastasis (21 cases) had obviously low bax expression (43%) and high bcl-2 expression (76%), showing significant difference to the group without lymph node metastasis (P<0.05). Conclusion: The antiapoptosis function of bcl-2 was stronger than bax in breast cancer. Protein bax and bcl-2 assay may be useful in understanding the biological behaviors of breast cancer.

THE OVEREXPRESSION OF APOPTOSIS-RELATED GENES OF P_53 AND BCL-2 IN CERVICAL CARCINOMA

Objective To investigate the significance of overexpression of p5s and bcl-2 protein in carcinogene- sis of cervix. Methods 10 cases of cervical intraepithelial neoplasis(CIN) and 57 cases of invasive cancer were in- vestigated with immunohistochemistry technique. Results The overexpresion or P53 protein ir CIN and cervical can- cer was significantly higher than that or control, respectively (P<0.01). But there was no significant difference be- tween CIN and cervical cancer(P>0.05). The immunoreactivity of bcl-2 in CIN was much more higher than that of control (P<0.05). The positive rate and immunoreactivity of bcl-2 in cervical carcinoma were both remarkably high- er than those of control (P<0.0l),but there was no significant difference between CIN and cervical carcinoma (P> 0.05). It was also found that there was a remarkably positive correlation between the overexpression of bcl-2 and P53 (P<0.01). Conclusion Because of the loss of wtP53 function,the expression of bcl-2 can not be down-reguated, which is associated with the pathogenesis and development of cervical carcinoma.

Baicalin extracted from Huangqin(Radix Scutellariae Baicalensis) induces apoptosis in gastric cancer cells by regulating B cell lymphoma(Bcl-2)/Bcl-2-associated X protein and activating caspase-3 and caspase-9

OBJECTIVE:To evaluate the effects of baicalin in human gastric cancer cells, including apoptosis-inducing effects, and to investigate its underlying mechanisms of action.METHODS:Cell proliferation and apoptosis assays were performed to investigate the anti-proliferation effects of baicalin in human gastric cancer BGC-823 and MGC-803 cells.Real time-quantitative polymerase chain reaction and Western blotting analysis were performed to elucidate the molecular mechanisms underlying the anti-tumor properties of baicalin.RESULTS:In BGC-823 and MGC-803 gastric cancer cells treated with 80, 120, and 160 μmol/L baicalin for 48 h, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay showed that baicalin significantly inhibited cell proliferation in a dose-dependent manner, while flow cytometric analysis demonstrated that baicalin could induce apoptosis, also in a dose-dependent manner.Moreover, baicalin up-regulated the expression of caspase-3, caspase-9, and B cell lymphoma(Bcl-2)-associated X protein and down-regulated the expression of Bcl-2 at both the m RNA and protein level.CONCLUSION:Baicalin has potential as a therapeutic agent for gastric cancer by inducing apoptosis in cancer cells.

Effect of Icariin on apoptosis and expression of Fas,Fas ligand,B cell lymphoma,and Bcl-2-associated X protein in CD4＋ T lymphocytes from patients with ankylosing spondylitis

OBJECTIVE:To investigate the effects of icariin on apoptosis and the expression of Fas, Fas ligand(Fas L), B cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) in CD4+ T lymphocytes from patients with ankylosing spondylitis.METHODS:Primary cultures of peripheral blood CD4+ T lymphocytes were established and treated with icariin at high, medium, and low doses(0.5,0.25, and 0.125 mg/mL).Sulfasalazine treated and helthy cells were used as controls.Apoptosis of treated cells was determined by flow cytometry.Reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays were used to determine the effects of icariin on the expression of Fas, Fas L, Bcl-2, and Bax.The activity of caspase 8 and caspase 3 was determined by a colorimetric assay.RESULTS:The m RNA and protein expression of Fas,and activity of caspase 8 and caspase 3 in CD4+ T lymphocytes were increased by icariin(P < 0.05).Conversely, the m RNA and protein expression of Bcl-2 was decreased(P < 0.05).The expression of Fas L and Bax were not significantly different between groups.The proapoptotic effects of icariin were dose-dependent.CONCLUSION:Icariin induces the apoptosis of CD4 + T cells from patients with AS comparing to normal control.Therefore, the induction of apoptosis may be the likely mechanism of action of icariin's antirheumatics activities.

BCL2、BCL6、MUM1蛋白表达及其与弥漫性大B细胞淋巴瘤预后的关系

目的探讨弥漫性大B细胞淋巴瘤(DLBCL)组织中MUM1、BCL2和BCL6蛋白的表达及其与DLBCL临床病理特征及预后的关系。方法收集山东省潍坊市人民医院2010年2月至2014年2月80例有完整临床和随访资料的新确诊DLBCL患者的病理组织标本,应用免疫组织化学法检测肿瘤组织中BCL2、BCL6和MUM1蛋白的表达,分析其相互关系及其与DLBCL临床病理特征及预后的关系。结果80例DLBCL患者肿瘤组织中BCL2、BCL6及MUM1 3种蛋白的阳性表达率分别为36.2%(29/80)、65.0%(52/80)、77.5%(62/80)。MUM1阳性组、阴性组患者平均生存期分别为35.4、51.7个月;BCL2阳性组、阴性组患者平均生存期分别为35.7、47.2个月;BCL6阳性组、阴性组患者平均生存期分别为54.5、35.1个月。MUM1阴性组、BCL6阳性组患者预后相对较好,差异有统计学意义(P<0.05),而BCL2与DLBCL患者预后的关系尚不确切。结论 MUM1、BCL2及BCL6蛋白表达情况为DLBCL的临床治疗和预后分析提供了重要信息,但目前对其作用机制尚缺乏了解,值得深入研究。

Ki-67与Bcl-2在非霍奇金淋巴瘤中的表达及其临床意义

目的 探讨增殖相关抗原Ki 6 7和抗凋亡蛋白Bcl 2的检测 ,观察其在非霍奇金淋巴瘤(NHL)中的表达及临床意义。方法 用免疫组织化学染色的方法 ,对 35例NHL外科活检石蜡包埋组织切片进行Ki 6 7抗原和Bcl 2蛋白的检测。结果 (1)Ki 6 7在低度恶性NHL组中的表达低于高度恶性组 (P <0 .0 1) ,而Bcl 2在低度恶性NHL组中的表达高于高度恶性组 (P <0 .0 1)。 (2 )B细胞性NHL中Bcl 2的表达高于T细胞性NHL(P <0 .0 2 ) ,而Ki 6 7表达差异无显著性 (P >0 .1)。 (3)首次化疗缓解组中的Bcl 2表达低于未缓解组 (P <0 .0 1) ,而Ki 6 7表达差异无显著性 (P >0 .1)。 (4)高Ki 6 7表达组的生存期比低表达组短 (P <0 .0 5 ) ,而Bcl 2的表达在两组之间无差异。结论 增殖相关抗原Ki 6 7和抗凋亡蛋白Bcl 2的表达与NHL的恶性程度和预后密切相关 ,是了解肿瘤发生和判断NHL预后的有效指标。

抗凋亡蛋白Bcl-2在非霍奇金淋巴瘤中的表达、分布及其临床意义

研究抗凋亡蛋白Ｂｃｌ－２在非霍奇金淋巴瘤（ＮＨＬ）中的表达、分布及其临床意义。方法采用４重ＰＡＰ免疫组织化学法，对比分析了１２例反应性淋巴滤泡增生与７１例ＮＨＬ的常规石蜡包埋淋巴结组织中Ｂｃｌ－２蛋白的表达与分布。结果（１）９３．０％Ｔ、Ｂ淋巴细胞性ＮＨＬＢｃｌ－２蛋白阳性。（２）８／９例滤泡中心起源的弥漫型ＮＨＬ中，Ｂｃｌ－２蛋白局限于中心细胞，而中心母细胞多为阴性。（３）５／１０例呈浆细胞样分化趋势的弥漫性大Ｂ细胞ＮＨＬ，Ｂｃｌ－２蛋白表达增强。（４）７例滤泡型ＮＨＬ中，Ｂｃｌ－２蛋白主要定位于瘤性滤泡中央，而其周围则相对稀疏。相反，生理性淋巴滤泡中，Ｂｃｌ－２蛋白则主要分布于套区，生发中心均为阴性。结论Ｔ、Ｂ淋巴细胞ＮＨＬ均能表达Ｂｃｌ－２；Ｂｃｌ－２表达可能与ＮＨＬ分化水平相关；Ｂｃｌ－２蛋白免疫组化染色有助于鉴别反应性淋巴滤泡增生过长与滤泡型淋巴瘤。

STAT3磷酸化对胃MALT淋巴瘤细胞凋亡的调控作用

目的 探讨STAT3磷酸化对胃MALT淋巴瘤细胞凋亡的调控作用。方法 采用SP免疫组织化学方法检测了 45例胃MALT淋巴瘤细胞及 13例滤泡型淋巴结反应性增生病 (RH)的p -STAT3、促凋亡基因 /蛋白fas和抗凋亡基因 /蛋白bcl-2的表达。结果 p -STAT3、fas及bcl-2蛋白在胃MALT淋巴瘤细胞中均呈过度表达 (阳性率分别为 64 .4%、60 .0 %和 66.7% ) ;低度恶性组的fas蛋白阳性信号强度明显低于高度恶性组 (P <0 .0 5 ) ,而bcl-2蛋白则明显高于高度恶性组 (P <0 .0 5 ) ,在低度与高度恶性组间的p -STAT3阳性表达强度无明显差异 (P >0 .0 5 ) ;p -STAT3与fas蛋白在胃MALT淋巴瘤细胞中的阳性表达呈明显的负相关 (r=-0 .6791,P <0 .0 5 ) ,而与bcl -2蛋白的阳性表达则呈明显的正相关 (r=0 .72 3 1,P <0 .0 5 )。结论 STAT3活化可能通过抑制细胞凋亡导致胃MALT淋巴瘤细胞的恶性转化及高增殖性 。

Alanyl-glutamine dipeptide inhibits hepatic ischemiareperfusion injury in rats

AIM： To investigate the protective effect and mechanism of alanyl-glutamine dipeptide （Ala-GIn） against hepatic ischemia-reperfusion injury in rats. METHODS： Rats were divided into group C as normal control Group （/7=16） and group G as alanyl-glutamine pretreatment 07=16）. Rats were intravenously infused with 0.9% saline solution in group C and Ala-GIn -enriched （2% glutamine） 0.9% saline solution in group G via central venous catheter for three days. Then all rats underwent hepatic warm ischemia for 30 min followed by different periods of reperfusion. Changes in biochemical parameters, the content of glutathione （GSH） and the activity of superoxide dismutase （SOD） in liver tissue, Bcl-2 and Bax protein expression and morphological changes of liver tissue were compared between both groups. RESULTS： One hour after reperfusion, the levels of liver enzymes in group G were significantly lower than those in group C （P〈0.05）. Twenty-four hours after reperfusion, the levels of liver enzymes in both groups were markedly recovered and the levels of liver enzyme in group G were also significantly lower than those in group C （P〈0.01）. One and 24 h after reperfusion, GSH content in group G was significantly higher than that in group C （P 〈0.05）. There was no statistical difference in activities of SOD between the two groups. One and 24 h after reperfusion, the positive expression rate of Bcl-2 protein was higher in group G than in group C （P〈0.05） and the positive expression rate of Bax protein was lower in group G than in group C （P〈0.05）. Histological and ultrastructural changes of liver tissue were inhibited in group C compared to group G. CONCLUSION： Our results suggest that Ala-GIn pretreatment provides the rat liver with significant tolerance to warm ischemia-reperfusion injury, which may be mediated partially by enhancing GSH content and regulating the expression of Bcl-2 and Bax proteins in the liver tissue.

胃肠道黏膜相关淋巴组织淋巴瘤中凋亡增殖相关蛋白的表达

目的 探讨胃肠道黏膜相关淋巴组织型淋巴瘤 (MALToma)中凋亡、增殖相关蛋白的表达情况。 方法 采用免疫组化法检测胃肠道 34例MALToma中PCNA和p5 3(SP法 ) ,Bcl 2和Caspase 3(CSA法 )的表达。 结果 Caspase 3和PCNA的表达与肿瘤恶性度有关 ,高恶组Caspase 3和PCNA的阳性表达均高于低恶组 (u =2 .5 15 ,P <0 .0 5 ;u =2 .74 5 ,P <0 .0 1)。Bcl 2、p5 3在不同恶性组中的表达无显著性差异。 结论 凋亡、增殖相关蛋白Caspase 3、PCNA可能参与了胃肠道黏膜相关淋巴组织型淋巴瘤的发病及恶性进展。

Inhibition of the Growth of Raji Cells by Precursor MicroRNA-15a

OBJECTIVE To investigate the effects of microRNA-15a （miR- 15a） on the growth of the lymphoma Raji cell line. METHODS A eukaryotic expression vector of precursor mLR-15a （pre-miR-15a） was constructed. Paired oligonucleotides for pre- miR-15a expression were designed and synthesized chemically. Annealed oligonucleotides were inserted into a pGCSIL- GFP vector under a U6 promoter to construct a pre-miR-15a expression plasmid. Oligonucleotides with a scrambled sequence which were used as a negative control were also constructed into the pGCSIL-GFP vector. A recombinant expression vector was identified through PCR and sequencing. A pre-miR-15a expression plasmid and a negative control plasmid were all transferred into Raji cells with lipofectamine 2000. Quantitative reverse-transcription polymerase chain reaction （qRT-PCR） was employed to explore expression of miR-15a. The expression levels of the protein were assayed via immunofluorescence. The growth effect of Raji cells was measured by CCK8 assay. Apoptosis was determined using Hoechst dyeing and flow cytometry. The growth-inhibitory effect on Raji cells was measured using a CCK8 assay. RESULTS The results showed that the insertion sequence was correct. The expression level of miR-15a was obviously higher in Raji cells transferred with pre-miR-15a plasmid than in the blank group and in the negative control group. After Raji cells were transferred with the pre-miR-15a expression plasmid for 48 h, Bcl-2 protein expression levels were obviously decreased, which indicated that there was a significant difference as compared with the negative control group and blank group （P 〈 0.05）. The CCK8 assay showed that transfection of pre-miR-15a expression plasmid decreased the cell growth at 24, 48 and 72 h post-transfection. After Raji cells were transferred with pre-miR-15a expression plasmid, apoptotic cells can be seen with Hoechst dyeing, and the cell apoptotic rate was obviously higher than that in blank group and negative control group. CONCLUSION Pre-miR-15a can induce apoptosis and inhibit the growth of Raji cells.