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Detection of Aneuploidy from Single Cells by Array-Comparative Genetic Hybridization

Jiawei LING ; Cong FANG ; Yanwen XU ; Guanglun ZHUANG

Journal of Sun Yat-sen University(Medical Sciences).2009;30(4):468-472,476.

[Objective] To set up an optimized protocol for aneuploidy detection from single cells through Array- Comparative Genetic Hybridization (CGH).[Method] Two cell lines,trisomy 18 (Tri-18;GM02732,47,XY,+18) and chromosome 4 segment deletion [sDel-4;GM00343,46,XY,4(del) (qter ＞ p14)],were used in the study.In combination of 10 k 2.0 SNP mapping array platform and multiple displacement amplification (MDA),the diagnostic accurate rates of MDA product from single cells of the two cell lines using gDNA and single-cell MDA product as reference were compared.[Result] An extremely lower call rate (3.2 ± 1.2)% in the negative control group was observed compared to the experiment groups.When the single-cell MDA product was used as reference,the standard deviations of Log2 (signal intensity ratio) were significantly decreased in both groups,compared with when the gDNA as reference (P ＝ 0.004).Through CNAT analytic software,some specific chromosomes (16,17,19,and 22) presented obvious preferential amplification (PA) when the gDNA was used as reference,but this PA could be eliminated when single-cell MDA product was used as reference.[Conclusion] 10 k 2.0 SNP mapping array in combination with MDA could be a quick,highly efficient and accurate method to detect aneuploidy in single cells.

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Screening for Mutations of MODY 1-5 Genes in Chinese Families with Early-onset Type 2 Diabetes

Jinhua YAN ; Yunfeng SHEN ; Qiuqiong YU ; Hua LIANG ; Mengyin CAI ; Jianping WENG

Journal of Sun Yat-sen University(Medical Sciences).2009;30(4):437-440,封3.

[Objective] The aim of our study was to seek the mutations in MODY1～5 genes in Chinese population by direct sequencing in probands from families with early-onset type 2 diabetes.[Methods] Variants screening in MODY 1-5 genes were performed by PCR and direct sequencing in 19 probands from early-onset type 2 diabetes families.[Results] We found no mutation but many polymorphisms.There were 6,5,15,1,and 1 variants in MODY 1-5 genes respectively.[Conclusion] Our negative results in MODY genes suggest the genetic heterogeneity of different populations.Mutations in MODY 1-5 genes might not be the cause of diabetes in those 19 families.

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Myoepithelial Carcinoma of Nasopharynx:Clinicopathologic Features and Relationship with Epstein-Barr Virus

Jin XIANG ; Jia FU ; Jiabin LU ; Qiuliang WU

Journal of Sun Yat-sen University(Medical Sciences).2010;31(2):265-268,273.

[Objective]To analyze the clinical and pathological characteristics of myoepithelial carcinoma of nasopharynx,as well as its relationship with Epstein-Barr virus.[Methods]We report twelve cases of myoepithelial carcinoma of nasopharynx,study its clinical,histopathologic,and immunohistologic features,follow-up data and EBERS in-situ hybridization.[Results]Among age of patients ranged from 24 to 65 years(mean age=46 years).Imagery data demonstrated an extensive nodosity filling the nasopharynx,Of these 12 cgses,myoepithelial markers such as S-100,SMA,P63,CK14,CK5/6 and epithelial markers such as CK,CKL,CKH were not always all expressed at one case,but at less one of these two kind of markers expressed at one cage.The results of EBERS in-situ hybridization of 12 cases were negative.[Conclusion]Myoepithelial carcinoma is a low malignat tumor.It seldom takes place in nasopharynx,dispite its morphologic heterogeneity it has some special morphologic and immuohistologic characteristics as well as special result of EBERS in-situ hybridization,which may support its diagnosis.The combinde radiotherapy and chemotherapy after surgical removal of the tumor will control the recurrence and metabasis effectively.

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Mechanism of Growth of Human Prostate Cancer Cells Inhibited by DATS

Jianwei HAO ; Shaopeng QIU ; Xiaopeng MAO ; Yu CHEN ; Shengjie GUO ; Bin HUANG

Journal of Sun Yat-sen University(Medical Sciences).2010;31(2):186-189,194.

[Objective]This study was designed to determine growth inhibition of diallyl trisulfide(DATS)in human prostate cancer cells by inducing apoptosis and further to investigate the mechanism underlying such effect.[Methods]Growth inhibition by DATS was estimated by the tetrazolium(MTr)assay.Apoptosis induction in DATS-treated cells was assessed by fluorescence microscopy analysis of cells with condensed and segmented nuclei following staining with DAPI and flow cytometric analysis of cells with sub-G1 DNA content following staining with propidium iodide.Protein levels of apoptosis regulating proteins were determined using western blot.The activity of caspase-3 was measured using a colorimetric assay.[Result]DATS showed tumor growth inhibition in a time-and dose-dependent manner,IC_(50) of DATS was 14 μmol/L at 72 h.DATS evoked apoptosis as confirmed by cell morphology and by the analysis of flow cytometry.The expression of Bcl-2 and Bcl-xL,the apoptosis-suppressing proteins,was more down-regulated.The activity of caspase-3 was enhanced by DATS.[Conclusion]DATS inhibits growth of prostate cancer cells by inducing apoptosis in association with down-regulation of Bcl-2 and Bcl-xL and activation of caspase-3.

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Effect of Cyclosporine A on Activity and Expression of MMP-2 and MMP-9 in Renal Tubular Epithelial Cells

Zhihui YUE ; Liangzhong SUN ; Yijuan LI ; Ying MO ; Xiaoyun JIANG ; Shumei CHEN

Journal of Sun Yat-sen University(Medical Sciences).2010;31(1):91-94,133.

[Objective] To observe the effect of cyclosporine A (CSA) on the activity and expression of MMP-2 and MMP-9 in renal tubular epithelial cells. [Methods] NRK52E cells were cultured until its reached confluent. Then NRK52E cells were exposed to different concentration of CSA (0, 0.42, 0.84, 4.2, and 8.4 μmoL/L) for 48 h or 72 h respectively. MMP-2 and MMP-9 activities were detected by gelatin zymography. The expression of MMP-2 and MMP-9 mRNA were detected by RT-PCR. [Results] MMP-2 activity and mRNA levels were decreased in a dose dependent manner after exposed to different concentration of CSA for 48 h or 72 h in NRK52E cells. Compared with control (CSA 0 μmoL/L), CSA 0.42, 0.84, 4.2, 8.4 μmoL/L decreased the MMP-2 activities to 27%, 24%, 11%, and 9% respectively; The differences are significant, P<0.05. But the MMP-9 activity and mRNA levels were increased after exposed to CSA for 48 h or 72 h in NRK52E cells. Compared with control group, CSA 4.2 μmoL/L exposure increased MMP-9 activity to 438% in 48 h, and 237% in 72 h; the differences are significant as well, P<0.05. [Conclusion] A dose-dependent decrease in the expression and activity of MMP-2, and the up-regulation of the expression and activity of MMP-9 by CSA in renal epithelial cells may related to CSA associated tubulointerstitial damage.

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Role of TGF-β Expressed by Biliary Epithelial Cells in Pathogenesis of Cholestatic Liver Disease

Yao HE ; Baili CHEN ; Rongping YANG ; Zhirong ZENG ; Ming REN

Journal of Sun Yat-sen University(Medical Sciences).2010;31(1):85-90,99.

[Objective] To investigate the change of liver histology, proliferation of BDEC, and expression of TGF-β in different stages of liver chelestusis. [Methods] ①Rat cholestatic livers were induced by common bile duct ligation (BDL) and separated into 3 groups, namely control group (DO), 7 days after BDL group (DT), and 18 days after BDL group (D18). ② Histological changes of livers in different groups were evaluated based on Knodell HAI score. ③ Real-time PCR was used to detect the expression of TGF-β in liver tissue and isolated BDEC in different groups. ④ Statistically analyzing the correlation between Knedell HAl score and the levels of TGF-β_1 mRNA. ⑤ In vitro study was performed to investigate the effect of TGF-β_1 on an immortalized mouse intrahepatic bililary epithelial cell line (mIBEC). [Results] (I) Knedell HAI score and the proliferation of intrahepatic bile ducts increased as the liver cholestasis aggravated. ② The levels of TGF-β_1, TGF-β_2, and TGF-β_3 mRNA were significantly up-regulated in liver tissues and BDEC as the liver cholestasis aggravated. ③ Positive correlation was found between Knedell fibrotic score and the levels of TGF-β_1 mRNA in liver tissues and BDEC(r=0.9376, P<0.05 and r=0.9682, P < 0.01). ④ In vitro study showed that TGF-β_1 inhibited the proliferation of mIBEC. [Conclusion] ① Liver injury, biliary proliferation, and the levels of TGF-β mRNA expression increased as liver cholestasis aggravated. ② The interaction of TGF-β_1 and BDEC plays an important role in the pathogenesis of BDL induced cholestatic liver disease. ④ Up-regulated expression of TGF-β_1 mRNA in the proliferated BDEC participates in the formation of BDL induced cholestatic liver fibrosis.

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Effects of Hirudin on Ventricular Arrhythmia after Acute Myocardial Infarction in Rats

Jun LIU ; Lilong TANG ; Xinxue LIAO ; Anli TANG ; Chong FENG

Journal of Sun Yat-sen University(Medical Sciences).2010;31(1):50-54,78.

[Objective] To determine the effects and possible mechanism of the thrombin antagonist r-RGD-Hirudin (HIR) on ventricular arrhythmia(VA) after acute myocardial infarction (AMI). [Methods] Seventy adult male Sprague-Dawley rats were randomly subjected to the 10 groups according to duration of left coronary occlusion: HIR 0 min, HIR 5 rain, HIR 10 min, HIR 20 min, HIR 30 min, and normal saline(NS) 0 min, NS 5 min, NS 10 min, NS 20 min, NS 30 min; and the average of every group is 7 rats. Acute myocardial infarction was produced by the occlusion of the left anterior descending coronary artery, then the measurements of arrhythmia and infarction sizing by Evans blue were assessed as well as the expression of three isoforms of inositol 1,4,5-trisphosphate receptors (IP3Rs) mRNA in isehemic myocardium by reverse transeriptase polymerase chain reactions (RT-PCR). [Results] Compared with NS groups, the measurements of VA in HIR were reduced significantly in 5 to 20 minutes after AMI (P<0.05). The incidence of VA was all positive related to the expression of three isoforms of IP3Rs mRNA (P<0.01). Compared with NS groups, the expression of type2,inositol 1,4,5-trisphosphate receptor (IP3R2) mRNA at 10 min and type3, inositol 1,4,5-trisphosphate receptor mRNA (IP3R3) at 10 min and 20 min after AMI were significant decreased (P<0.05) in HIR groups. [Conclusion] The thrombin antagonist r-RGD-Hirudin exerts its myocardial protection against ventricular arrhythmia after acute myocardial infarction possible through IP3R2 and IP3R3 and not typel, inositol 1,4,5-trisphosphate receptor (IP3R1).

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Molecular Mechanisms and Interference Strategies of Glaucoma and Myopia

Jian GE

Journal of Sun Yat-sen University(Medical Sciences).2010;31(2):154-160,165.

The paper briefly reports the preliminary research results,focusing on the pathogenesis,mechanisms and interference strategies of glaucoma and myopia.(1)The myocilin gene has been found a mutation in Pro370Leu in GZ.1 and PN.1 glaucoma pedigrees,and the theory of protein misfolding and mitochondria dysfunction in glaucoma was then firefly elucidated.We further explored the modulation of wound healing by siRNA,neuro-protection by targeting drugs with drug deliver system and stem cells/iPS in glaucoma therapy,which offer a revolutionary strategy for glaucoma:differential treatment for specific stages.(2)Natural myopia model was successfully established in rhesus monkeys and retinal mechanisms and central nervous system were both found to play an important role during myopia development,and a variety of new optical interventions were therefore employed on human beings by series of randomized,controlled,double-masked prospective clinical trials.All these preliminary results offer a new insight into the new model for the diagnosis and treatment of glaucoma and myopia.

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Clinical Significance of Approximated Molecular Subtypes of Invasive Breast Cancer

Qirong GENG ; Donggeng LIU ; Yanxia SHI ; Jin XIANG ; Yue LU

Journal of Sun Yat-sen University(Medical Sciences).2009;30(4):458-462,467.

[Objective] To investigate the clinical significance of approximated molecular subtype.[Methods] Four different subtypes were defined by immunohistochemical (IHC) markers as estrogen receptor (ER),progesterone receptor (PR) and HER2.Clinical characteristics as distribution,age,menopausal status,histopathologic type and grading,tumor size,lymph node status,time to and place of first tumor recurrence were analyzed.[Results] Among all 703 cases,Luminal A was the majority (51.5%) and the least were Luminal B and HER2+.90.8% the patients were less than 59 years old with Luminal A.Different subtype has different histopathologic grading.Luminal A was more than others among grading 1 tumors and it has more T1 tumors,while T4 tumors was most among HER2+ ones.Lymph nodes metastasis has no significant difference between different groups,other than high proportion tendency of N3 stage in Luminal B and HER2+.In addition,Luminal A has the longest time to the first recurrence while HER2+ was the shortest.Moreover,HER2+ and Basal-like have high rates of lung metastasis.Different from foreign reports,the distribution of the first recurrent place and distant metastases have no significant difference.[Conclusion] The classification of breast cancer approximated molecular subtype defined by IHC markers was feasible.The distribution is similar to other countries.Luminal A has rather better clinical characteristics while HER2+ and Basal-like have high rates of lung metastasis.However,Luminal A has the highest proportion of young patients,which is different from other countries.

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Inhibitory Effect of CTLA-4Ig Fusion Protein on Atherosclerosis in ApoE-/- Mice

Jie CHEN ; Yujie LI ; Dongdan ZHENG ; Yan XIONG ; Xin LI ; Xiaoxing LIAO

Journal of Sun Yat-sen University(Medical Sciences).2009;30(4):395-399,417.

[Objective] To investigate the inhibitory effect of CTLA-4Ig fusion protein on atherosclerosis in the mice with an apolipoprotein-E gene defect fed on cholesterol diet.[Methods] Twenty-five male 10-week-old ApoE-/- mice were selected and fed on cholesterol diet for 4 weeks,5 out of which were executed at random as control group and their pathological sections were kept to observe the early fatty streaks.The other 20,divided into CTLA-4Ig treatment group,PBS group,IgG1 group,and blank group at random,5 in each.Three groups were given intraperitoneal injection of CTLA-4Ig (10 μg per time),PBS (100 μL per time),Rat-IgG1 (10 μg per time) respectively,twice a week,for 8 weeks.The blank group has no treatment.Followed by 8-week treatment,the whole aorta from the root to crotch of iliac artery was separated after anesthesia with the intraperitoneal injection of 1 % pentobarbital.Subsequently,the area ratio of plaque and lumen,the thickness ratio of endangium and tunica media,the lipid-soaking extent intra-plaque and the content of collagen fibrils and smooth muscle cells intra-plaque were analyzed by image-processing soft.[Results] After fed on cholesterol diet for 4 weeks,there were obviously atherosclerosis in the aorta in the ApoE-/- mice.There were typical atherosclerotic plaque in ApoE-/- mice fed on cholesterol diet after another 8 weeks.The area ratios of plaque and lumen in CTLA-4Ig group,PBS group,IgG1 group,and blank group were 0.27 ± 0.08,0.40 ± 0.08,0.43 ± 0.08,and 0.46 ± 0.10,and obviously increased than those in control group (0.05 ± 0.01,P ＜ 0.05).The thickness ratios of endangium and tunica media in four groups were 2.6 ± 0.6,6.0 ± 0.9,5.7 ± 0.8,and 5.9 ± 0.6 and obviously increased than those in control group (0.5 ± 0.1,P ＜ 0.05).The lipid-soaking extent intra-plaque in experimental groups were 26.0 ± 3.0,40.8 ± 5.7,40.6 ± 3.0,and 43.2 ± 5.7,and were obviously increased than those in control group (7.2 ± 1.4,P ＜ 0.05 ).It was found that the area ratio of plaque and lumen,the thickness ratio of endangium and tunica media,and the lipid-soaking extent intra-plaque in CTLA-4Ig group were significantly lower than those in PBS group,IgG1 group,and blank group (P ＜ 0.05),but there was no significant difference in those between the PBS group,IgG1 group,and blank group (P ＞ 0.05).The content of collagen fibrils in CTLA-4Ig group were 16.0 ± 1.1 and higher than those in PBS group,IgG1 group,and blank group (8.6 ± 1.2,9.2 ± 1.5,and 9.0 ± 1.3,P ＜ 0.05).The content of smooth muscle cells in plaque in CTLA-4Ig group were 11.8 ± 1.0 and higher than those in PBS group,IgG1 group,and blank group (7.8 ± 0.8,7.5 ± 0.9,and 7.3 ± 0.7,P ＜ 0.05).There was no significant difference in content of collagen fibrils and smooth muscle cells between the PBS group,IgG1 group,and blank group (all P ＞ 0.05).[Conclusion] CTLA-4Ig fusion protein could evidently inhibit the atherosclerosis progression and enhance the stability of plaque through increasing the content of collagen fibrils produced by smooth muscle cells intra-plaque in ApoE-/- mice fed on cholesterol diet.

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