PCR-SSCP-DNA sequencing method in detecting PTEN gene mutation and its signifi cance in human gastric cancer

AIM: To discuss the possible effect of PTEN gene mutations on occurrence and development of gastric cancer. METHODS: Fifty-three gastric cancer specimens were selected to probe PTEN gene mutations in genome of gastric cancer and paracancerous tissues using PCR-SSCP-DNA sequencing method based on microdissection and to observe the protein expression by immunohistochemistry technique. RESULTS: PCR-SSCP-DNA sequencing indicated that 4 kinds of mutation sites were found in 5 of 53 gastric cancer specimens. One kind of mutation was found in exons. AA-TCC mutation was located at 40bp upstream of 3’ lateral exon 7 (115946 AA-TCC). Such mutations led to terminator formation in the 297th codon of the PTEN gene. The other 3 kinds of mutation were found in introns,including a G-C point mutation at 91 bp upstream of 5’ lateral exon 5(90896 G-C),a T-G point mutation at 24 bp upstream of 5’ lateral exon 5 (90963 T-G),and a single base A mutation at 7 bp upstream of 5’ lateral exon 5 (90980 A del). The PTEN protein expression in gastric cancer and paracancerous tissues detected using immunohistochemistry technique indicated that the total positive rate of PTEN protein expression was 66% in gastric cancer tissue,which was significantly lower than that (100%) in paracancerous tissues (P < 0.005). CONCLUSION: PTEN gene mutation and expression may play an important role in the occurrence and development of gastric cancer.

EFFECTS OF MUTATION AND EXPRESSION OF PTEN GENEmRNA ON TUMORIGENESIS AND PROGRESSION OFEPITHELIAL OVARIAN CANCER

Objective To investigate the mutation and expression of tumor suppressor gene-PTEN mRNA and explore their roles in tumorigenesis and progression of ovarian cancer. Methods Mutated exon 5 of PTEN gene was examined in normal ovary(n = 5), ovarian cyst (n =5), ovarian borderline tumor (n = 9), epithelial ovarian cancer(n = 60), and ovarian cancer cell line (n = 1)by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). mRNA expression of PTEN gene was evaluated in corresponding tissues and cell line by reverse transcription polymerase chain reaction(RT-PCR). The mutation and mRNA expression of PTEN gene were compared with clini-copathological features of ovarian cancer. Results Mutated exon 5 of PTEN gene was detected only in 5(7.1%)cases of epithelial ovarian cancer. mRNA expression level of PTEN gene in ovarian borderline tumor or ovarian cancer was lower than that in normal ovary or ovarian cyst(P < 0.05). The level of PTEN gene mRNA expression was negatively correlated with clinicopathological staging of ovarian cancer, whereas positively correlated with histological differentiation (P < 0.05). mRNA expression level of PTEN gene in ovarian endometrioid cancer was significantly lower than that in ovarian serous or mucinous cancer (P < 0.05=. Conclusions Mutation of PTEN gene occurs in ovarian cancer. Down-regulated expression of PTEN is probably an important molecular event in tumorigenesis of ovarian cancer. Abnormal expression of PTEN gene is involved in progression of ovarian cancer. Reduced expression of PTEN gene is closely associated with tumorigenesis and pathobiological behaviors of ovarian endometrioid cancer.

Reversal of Multidrug Resistance and Inhibition of Phosphorylation of AKT in Human Ovarian Cancer Cell Line by Wild-type PTEN Gene

The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C 13K cells were 2.04 ± 0.10, 0.94± 0.04 respectively and the expression of p-Akt protein （ 0.94± 0.07） was lower than those in control groups （1.68 ±0.14, 1.66± 0.10） （P〈 0.05）. The IC50 of DDP to C 13 K cells transfected with PTEN （7.2± 0.3 la mol/L） was obviously lower than those of empty-vector transfected cells and non-transfected cells （12.7±0.4 lamol/1, 13.0±0.3 lamol/L） （P〈0.05）. The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were （41.65___0.87）%, （18.61 ±0.70）% and （15.28±0.80）% respectively, and the difference was statistically significant （P〈0.05）. PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C 13K with multidrug-resistance by decreasing the expression of p-Akt.

Relationship between promoter methylation and mRNA expression of PTEN gene and gastric carcinoma

Objective:To probe into the relationships between PTEN gene expression,the promoter methylation and gastric cancer and its clinical pathological specific features.Methods:We analyzed the PTEN gene promoter methylation and mRNA expression status in gastric cancer tissues and its adjacent normal tissues by methylation specific PCR(MSP) and reverse transcription-polymerase chain reaction(RT-PCR) techniques.Results:PTEN promoters in 48.2%(27/56) gastric cancer tissues and 3.6(2/56) adjacent normal tissues were methylated and the PTEN promoter methylation rate in carcinoma tissues was obviously higher(P < 0.05).Of the 2 cases where the adjacent gastric tissues were methylated,the gastric cancer tissues were both methylated.Of the 29 gastric cancers with lymph node metastasis,19 had their PTEN gene promoters methylated and the PTEN gene promoter methylation in cases with lymph node metastasis was obviously higher than that without lymph node metastasis(P < 0.05).RT-PCR result showed that no expression of PTEN mRNA existed in any of the methylated gastric cancer tissues.Conclusion:The expression loss of PTEN gene mRNA in gastric cancers is related to their promoter methylation and might be one of the reasons for the generation,development and metastasis of gastric cancers.

DOWN-REGULATED EXPRESSION OF PTEN GENE AND LOH OF ITS EPIGENETIC MICROSATELLITES IN GASTRIC CARCINOMA

Objective.To investigate PTEN expression and loss of heterozygosity(LOH)of its epigenetic microsatel-lites in gastric carcinoma and explore their roles in tumorigenesis and progression of gastric carcinoma.Methods.LOH of epigenetic microsatellites of PTEN(D10S541,D10S583and D10S1687)was exam-ined in advanced gastric carcinomas(n=56)by PCR-SSCP.The mRNA and protein expressions of PTEN gene were evaluated in normal mucosa(n=56),early(n=11)and advanced carcinomas(n=56)of the stomach using RT?PCR and immunohistochemoistry respectively.PTEN mRNA and protein expressions were compared with clinicopathological staging and lymph node metastasis of tumors.The relationship be-tween PTEN mRNA expression and LOH of microsatellites was discussed,as well as relationship between PTEN mRNA and protein expression.Results.LOH of D10S541,D10S583and D10S1687was found in28.6%(16/56)of advanced gas-tric carcinomas.The positive rates of PTEN expression were80.4%(45/56),45.5%(5/11)and32.1%(18/56)in normal gastric mucosa,early and advanced gastric carcinomas at mRNA level,while78.6%(44/56),36.4%(4/11)and28.6%(16/56)at protein level.PTEN mRNA and protein were less fre-quently expressed in early and advanced gastric carcinomas than normal gastric mucosa(P<0.05).There was negative correlation between PTEN mRNA expression and LOH of microsatellites(P<0.05).PTEN protein expression paralleled to its mRNA expression(P<0.05).The PTEN mRNA and protein expres-sions were negatively correlated with lymph node metastasis of advanced gastric carcinomas(P<0.05).Conclusion.Down?regulated expression of PTEN and frequent LOH of its epigenetic microsatellites might play an important role in gastric carcinogenesis.Reduced PTEN mRNA expression was closely as-sociated with LOH of its epigenetic microsatellites.Altered expression of PTEN might contribute to lymph node metastasis of gastric carcinoma by decreasing cell adhesion and apoptosis,increasing angiogenesis and cell mobility.

Expression and clinical significance of MN1 and PTEN gene in patients with acute myeloid leukemia

Objective:The aim of our study was to explore the correlations between both the genes,evolution and prognosis of the disease by detecting the expression of MN1(meningioma 1) gene and PTEN(phosphatase and tensin homolog) gene in patients with acute myeloid leukemia(AML).Method:MN1 and PTEN mRNA were detected by reverse transcription-PCR in mononuclear cells of 38 patients with AML and 13 patients with normal bone marrow.Results:Positive rates of MN1 and PTEN genes were 76.3% and 60.5% respectively in bone marrow mononuclear cells.The expression level of MN1 mRNA for the de novo group increased comparing with that for normal control group(P < 0.05),the expression level of PTEN mRNA for de novo group decreased obviously comparing with that for normal control group(P < 0.01),MN1 mRNA level decreased in remission group,while PTEN mRNA level increased comparing with that in de novo group(P < 0.05).MN1 mRNA level increased and PTEN mRNA decreased in relapsed group comparing with that in control group,and their difference was statistical significance(P < 0.05).The two genes' levels had negative correlations in acute myeloid leukemia(r =-0.314,P < 0.05).Conclusion:There is close correlations between expression of MN1 and PTEN genes and the prognosis and occurrence of acute myeloid leukemia,and their expression can be taken as significant indexes to access the de novo,relapse and prognosis of acute myeloid leukemia.

The establishment of stable transfection of human breast cancer cell line MDA-MD-468 with exogenous PTEN gene

To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly metastatic breast cancer cell line MDA-MD-468.After transfection,the cells were selected by G418.The resistant clones were chosen and expanded in DMEM culture medium.RT-PCR,immunohistochemical method and western blot were used to determine the expression of target genes.Results An anti-G418 cell clone was established and expanded in culture.The transfected PTEN gene MDA-MD-468 cells showed expression of PTEN mRNA and PTEN protein.Conclusion Human breast cancer cell line MDA-MB-468 established in this study expresses consistently exogenous PTEN genes.4 refs,6 figs.

The effects of antisense PTEN gene transfection on the growth and invasion of glioma cells

Objective:To study the effects of antisense PTEN gene on the growth and invasion of glioma cells. Methods:A pcDNA3. 1/Hygro (-) recombinant plasmid containing antisense PTEN gene fragment was constructed. Glioma cells of primary culture were transfected with antisense PTEN gene vector and stably transfected clones were selected. Then, the different growth and invasion abilities and the different MMP9 mRNA expressions of three kinds of cells were observed, including the transfected cells, untransfected cells and the cells transfected with empty vector. Results :The abilities of growth and invasion of the transfected cells and the expressions of MMP9 mRNA were obviously enhanced. Conclusion: Antisense PTEN gene could have a negative impact on the growth and invasion of primary culture glioma cells.

长链非编码RNA PTENP1通过miR-3611/PTEN基因途径调控宫颈癌进程的分子机制研究

目的 探讨长链非编码RNA PTENP1 (以下简称“PTENP1”)在宫颈癌细胞增殖、迁移和侵袭的分子机制。方法 选取2019年1月至2022年12月海南省妇女儿童医学中心诊断的54例的癌组织与癌旁组织,另选取宫颈癌细胞系(HeLa、SiHa、C33A、Caski)和正常宫颈上皮细胞系(H8),采用实时荧光定量PCR、蛋白质印迹法检测PTENP1、miR-3611、PTEN基因、PTEN蛋白水平。选取合适的宫颈癌细胞系,比较PTENP1在细胞质和细胞核中的表达情况,并进一步将其分为空白组(无处理)、pcDNA3.1组(转染pcDNA3.1)、pcDNA3.1-PTENP1组(转染pcDNA3.1-PTENP1)、NC组(阴性对照,转染模拟物或抑制剂NC)、miR-3611抑制剂组(转染miR-3611抑制剂)、pcDNA3.1-PTENP1+NC组(转染pcDNA3.1-PTENP1和NC)和pcDNA3.1-PTENP1+miR-3611 mimics组(转染pcDNA3.1-PTENP1和miR-3611模拟物)。比较空白组、pcDNA3.1组、pcDNA3.1-PTENP1组PTENP1、miR-3611、PTEN基因、PTEN蛋白及上皮间质转化的相关指标,采用细胞活力检测及流式细胞仪检测细胞增殖和凋亡情况;比较空白组、NC组、miR-3611抑制剂组miR-3611、PTEN基因、PTENP1水平;比较pcDNA3.1-PTENP1+NC组和pcDNA3.1-PTENP1+miR-361 1 mimics组的细胞增殖情况及上皮间质转化的相关指标。采用双萤光素酶报告基因及RNA下拉实验验证miR-3611与PTENP1、PTEN基因的靶向关系。结果 癌组织PTENP1、PTEN基因、PTEN蛋白水平低于癌旁组织,miR-3611水平高于癌旁组织(P<0.05)。HeLa、SiHa、C33A、Caski细胞PTENP1、PTEN基因、PTEN蛋白水平低于H8细胞,miR-3611水平高于H8细胞(P<0.05),后续实验选择HeLa、Caski细胞进行,PTENP1在HeLa、Caski细胞质中高表达。pcDNA3.1-PTENP1组PTENP1、凋亡率、上皮钙黏素、PTEN基因高于空白组,细胞增殖活力(培养48、72、96h)及ZEB1、Snail、波性蛋白、miR-3611低于空白组(P<0.05)。miR-3611抑制剂组miR-3611低于空白组,PTEN基因、PTENP1高于空白组(P<0.05)。pcDNA3.1-PTENP1+miR-361 1 mimics组细胞增殖活力(培养48、72、96 h)、上皮钙黏素低于pcDNA3.1-PTENP1+NC组,ZEB1、Snail、波性蛋白高于pcDNA3.1-PTENP1+NC组(P<0.05)。野生型miR-3611转染生物素标记的HeLa、Caski细胞的PTENP1水平高于转染生物素标记的空白细胞和突变型miR-3611转染生物素标记的细胞,分别转染PTENP1或PTEN野生型和miR-3611模拟物的293T细胞的相对萤光活性低于仅转染PTENP1或PTEN野生型的293T细胞(P<0.05)。结论 PTENP1通过竞争性结合miR-3611调控PTEN表达影响宫颈癌细胞增殖、迁移和侵袭。

Loss of heterozygosity on 10q23.3 and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions

AIM: To investigate the loss of heterozygosity (LOH) and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions. METHODS: Thirty cases of normal gastric mucosa, advanced and early stage gastric cancer, intestinal metaplasia, atrophic gastritis, and atypical hyperplasia were analyzed for PTEN LOH and mutations within the entire coding region of PTEN gene by PCR-SSCP denaturing PAGE gel electrophoresis, and PTEN mutation was detected by PCR-SSCP sequencing followed by silver staining. RESULTS: LOH rate found in respectively atrophic gastritis was 10% (3/30), intestinal metaplasia 10% (3/30), atypical hyperplasia 13.3% (4/30), early stage gastric cancer 20% (6/30), and advanced stage gastric cancer 33.3% (9/30), None of the precancerous lesions and early stage gastric cancer showed PTEN mutations, but 10% (3/30) of the advanced stage gastric cancers, which were all positive for LOH, showed PTEN mutation. CONCLUSION: LOH of PTEN gene appears in precancerous lesions, and PTEN mutations are restricted to advanced gastric cancer, LOH and mutation of PTEN gene are closely related to the infiltration and metastasis of gastric cancer.

mRNA EXPRESSION OF PTEN AND VEGF GENES IN EPITHELIAL OVARIAN CANCER

Objective:To investigate the mRNA expression of PTEN and vascular endothelial growth factor (VEGF) genes in ovarian cancer. Methods:We examined mRNA expression of PTEN and VEGF165 in normal ovary (n=5), ovarian cyst (n=5), ovarian borderline tumor (n=9), epithelial ovarian cancer (n=60) and ovarian cancer cell line (CAOV-3) by RT-PCR. Their expressions were compared with clinicopathological features of ovarian cancer. The relationship between their expressions was concerned in all ovarian samples as well. Results:mRNA expression level of PTEN gene was significantly lower in ovarian borderline tumor or ovarian cancer than that in normal ovary or ovarian cyst(P<0.05). It was negatively correlated with clinicopathological staging(P<0.05),whereas positively with histological differentiation (P<0.05). mRNA expression level of PTEN gene was significantly lower in ovarian endometrioid cancer than ovarian serous or mucinous cancer(P<0.05). mRNA expression level of VEGF165 gene was significantly higher in ovarian cancer than that in normal ovary or ovarian cyst(P<0.05). It was positively correlated with clinicopathological staging(P<0.05), whereas negatively with histological differentiation (P<0.05). mRNA expression level of VEGF165 gene was significantly higher in ovarian serous cancer than in other ovarian epithelial cancers (P<0.05). mRNA expression of VEGF165 gene was inversely correlated with mRNA expression level of PTEN gene. Conclusion:Down-regulated expression of PTEN and up-regulated expression of VEGF were considered as two important events in tumorigenesis of ovarian cancer and could be used as molecular markers to indicate the pathobiological behaviors of ovarian cancer. Decreased PTEN expression and increased VEGF expression were closely associated with tumorigenesis and pathobiological behaviors of ovarian endometrioid and serous cancer respectively. Reduced expression of PTEN gene might be involved in carcinogenesis and progression of ovarian cancer by up-regulating the VEGF expression to enhance angiogenesis.

一例PTEN新生杂合突变患儿临床表型与免疫特征分析

目的探讨一例PTEN杂合突变患儿的临床表型及免疫特征,丰富PTEN突变相关临床表型谱。方法收集患儿门诊及住院期间的病史资料、生化检查及影像学检查结果,抽取外周静脉血进行医学全外显子组综合检测,Sanger测序验证患儿及其父母PTEN基因突变位点,采用流式细胞术进行T细胞PI3K/Akt/mTOR通路磷酸化水平及T细胞亚群与其耗竭相关表面分子检测,采用蛋白质印迹法检测外周血单个核细胞PTEN蛋白表达水平,健康对照为患儿父亲。结果患儿以大头畸形(头围>P99)、疣状表皮痣、精神运动发育迟缓、学语延迟为主要临床表现,PTEN基因(NM_000314.8)c.388C>T(p.R130X)新生杂合突变,PTEN蛋白表达减少,血清IgA水平稍低(0.177 g/L),精细免疫分型CD4^(+)终末分化效应记忆T细胞、CD8^(+)终末分化效应记忆T细胞、过渡性B细胞比例及绝对数均增加,但T细胞PI3K/Akt/mTOR通路磷酸化水平正常。患儿以PTEN错构瘤综合征相关表型为主要表现,无明显PI3Kδ过度活化综合征样表型。结论该患儿PTEN基因的突变位点为国内首例,有助于丰富临床医生对该疾病的认识,提高诊治水平。

PTEN基因对肺癌作用及治疗的应用研究进展

PTEN基因是肺癌重要的抑癌基因,通过抑制PIP3生成,从而抑制AKT、mTOR活化,一方面直接抑制肿瘤细胞,另一方面减弱肿瘤间质对肿瘤细胞支持作用,抑制肺癌的发生、生长和转移。PTEN基因表达下降引起肺癌治疗耐药的发生,并反映患者的预后。然而因表观遗传和翻译后调控的存在,PTEN蛋白缺失明显高于PTEN基因突变发生率,因此相比于靶向PTEN基因的治疗药物,通过提高恶性肿瘤的PTEN蛋白表达可能是针对PTEN基因治疗肺癌及抑制耐药的一种更有前景的方法。

PTEN突变对黏膜黑色素瘤患者临床病理特征及预后的影响

目的探讨磷酸酶及张力蛋白同源物PTEN突变对黏膜黑色素瘤患者临床病理特征及预后的影响。方法收集2019年4月至2022年3月北京大学肿瘤医院黑色素瘤与肉瘤内科收治的66例黏膜黑色素瘤患者的临床信息和随访资料,采用二代测序技术检测PTEN基因的突变情况,分析PTEN突变与患者临床病理特征及预后的关系。结果66例黏膜黑色素瘤患者中,PTEN突变率为19.4%,PTEN突变状态与确诊时年龄和原发灶部位显著相关(P<0.05);在42例接受免疫治疗的患者队列中,PTEN突变患者的中位无进展生存期(mPFS)为3.37个月,显著低于野生型患者(9.2个月)(P<0.05)。多因素Cox回归分析进一步证实PTEN突变是影响免疫治疗疗效的独立预后因素(P<0.05)。结论PTEN在确诊时年龄小于60岁和原发灶为消化道黏膜的患者中突变频率更高,且PTEN突变是患者接受免疫治疗预后较差的独立预测因素。

卵巢癌中非编码RNA对PTEN基因的调控机制

卵巢癌(ovarian cancer,OC)是女性常见肿瘤,在女性中发病率与死亡率均为第八位、死亡率仅次于宫颈癌[1],亟需更加有效的治疗方式。OC的发生与PTEN表达水平异常有较强关联;OC有多种组织分型,伴随不同类型的PTEN表达异常,其中,PTEN表达水平下调在子宫内膜样亚型中最常见,而PTEN的拷贝丢失多见于高级别浆液性亚型[2]。在肿瘤细胞中,PTEN表达水平受到微小RNA(microRNA,miRNA)的直接调节[3-6],而miRNA又受其他非编码RNA(noncoding RNA,ncRNA)调控。因此,ncRNA相关的PTEN表达水平异常可能是OC的重要发病机制,分析、总结该机制的研究结果有助于全面了解OC病因,进而为其诊断和治疗提供新的思路。

子宫内膜癌PTEN基因968delA突变检测技术平台的构建及初步应用

目的 构建子宫内膜癌PTEN基因968delA突变检测技术平台,为临床分析PTEN基因968delA突变与子宫内膜癌的相关性提供技术支持。方法 设计PTEN基因968delA位点的特异性硫化修饰引物,以本实验室前期构建的包含PTEN基因968delA位点的突变型质粒和野生型质粒为模板,进行高保真聚合酶介导的双向引物延伸反应,采用正交设计进行PCR条件优化,再将其优化后采用突变敏感性分子开关技术检测从南华大学附属第一医院收集的62例临床患者子宫内膜组织样本PTEN基因968delA位点突变。结果 突变敏感性分子开关技术检测PTEN基因968delA位点最佳PCR反应条件为:30个循环、模板DNA(10 ng/μL)0.3μL、引物(10μmol/L)0.5μL、退火温度61℃,灵敏度达10^(-3)ng/μL。62例子宫内膜癌组织与21例正常子宫内膜组织标本均未发现PTEN基因968delA突变。结论 成功构建了子宫内膜癌PTEN基因968delA突变检测技术平台,为该技术在PTEN基因968delA的突变筛查中的应用提供了依据。

RNA激活上调PTEN基因表达对膀胱癌细胞增殖和凋亡影响的研究

目的探讨通过RNA激活(RNAa)技术上调PTEN基因的表达对人膀胱癌BIU-87细胞增殖和凋亡的影响。方法设计靶向抑癌基因PTEN启动子DNA序列互补的双链RNA分子(dsPTEN),转染入BIU-87细胞中,采用RT-PCR法及Western Blot检测PTEN基因mRNA及蛋白质的表达变化,MTT法检测BIU-87细胞增殖速度,流式细胞仪检测PTEN细胞凋亡率。结果dsPTEN转染BIU-87细胞后能显著上调PTEN基因的mRNA和蛋白的表达,与空白对照组、阴性对照组相比,经dsPTEN作用的BIU-87膀胱癌细胞增殖受到明显抑制,凋亡率较对照组明显升高(P<0.05)。结论RNAa技术能有效激活PTEN基因,使其表达上调并抑制膀胱癌BIU-87细胞增殖,诱导细胞凋亡,为膀胱癌的基因治疗提供了新的依据和方法。

PTEN蛋白及P53基因在结直肠癌中的表达

目的 探讨PTEN基因及P53基因在结直肠癌中的表达与分布,分析其临床意义。方法 收集59例结直肠癌和28例结直肠良性病变组织,采用免疫组织化学SABC染色法,染色观察PTEN及P53基因的表达和分布。结果 PTEN基因及P53基因在结直肠癌组织中的表达,低分化高于中高分化和良性病变组织及癌前病变组织。阳性表达平均光密度值(AOD)PTEN为(0.512±0.016),P53为(0.326±0.028);在结直肠良性病变组织中,PTEN基因及P53基因的平均光密度值则分别为(0.438±0.047)及(0.251±0.018)。差异均有统计学意义(P<0.05)。患者的年龄、性别、Ducks分期与PTEN基因及P53基因的表达水平无关,但二者基因的表达水平与恶性肿瘤的分化程度有关。结论 结直肠癌的分化程度越低,PTEN基因和P53基因表达越高。可作为结直肠癌肿瘤转移及预后监测的一项重要指标。

PTEN knockdown with the Y444F mutant AAV2 vector promotes axonal regeneration in the adult optic nerve

The lack of axonal regeneration is the major cause of vision loss after optic nerve injury in adult mammals. Activating the PI3K/AKT/mTOR signaling pathway has been shown to enhance the intrinsic growth capacity of neurons and to facilitate axonal regeneration in the central nervous system after injury. The deletion of the mTOR negative regulator phosphatase and tensin homolog （PTEN） enhances regeneration of adult corticospinal neurons and ganglion cells. In the present study, we used a tyrosine-mutated （Y444F） AAV2 vector to efficiently express a short hairpin RNA （shRNA） for silencing PTEN expression in retinal ganglion cells. We evaluated cell survival and axonal regeneration in a rat model of optic nerve axotomy. The rats received an intravitreal injection of wildtype AAV2 or Y444F mutant AAV2 （both carrying shRNA to PTEN） 4 weeks before optic nerve axotomy. Compared with the wildtype AAV2 vector, the Y444F mutant AAV2 vector enhanced retinal ganglia cell survival and stimulated axonal regeneration to a greater extent 6 weeks after axotomy. Moreover,post-axotomy injection of the Y444F AAV2 vector expressing the shRNA to PTEN rescued ～19% of retinal ganglion cells and induced axons to regenerate near to the optic chiasm. Taken together, our results demonstrate that PTEN knockdown with the Y444F AAV2 vector promotes retinal ganglion cell survival and stimulates long-distance axonal regeneration after optic nerve axotomy. Therefore, the Y444F AAV2 vector might be a promising gene therapy tool for treating optic nerve injury.