Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-ΔSHP-1 Antibodies

This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1（designated ΔSHP-1） and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 was amplified by PCR and then cloned into the pT7 expression vector. The recombinant pT7-ΔSHP-1 plasmid was used to transform Rosetta（DE3） E. coli cells. ΔSHP-1 was distributed in the exclusion body of E. coli cell extracts and was purified through a two-column chromatographic procedure. The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns. It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases. To generate polyclonal anti-ΔSHP-1 antibodies, purified recombinant ΔSHP-1 was used to immunize a rabbit. The resultant anti-serum was subjected to purification on ΔSHP-1 antigen affinity chromatography. The purified polyclonal antibody displayed a high sensitivity and specificity toward ΔSHP-1. This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.

Protein tyrosine phosphatase 1B regulates migration of ARPE-19 cells through EGFR/ERK signaling pathway

AIMTo evaluate whether protein tyrosine phosphatase 1B (PTP1B) contributed to initiate human retinal pigment epithelium cells (A)-19 migration and investigate the signaling pathways involved in this process.METHODSARPE-19 cells were cultured and treated with the siRNA-PTP1B. Expression of PTP1B was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). AG1478 [a selective inhibitor of epidermal growth factor receptor (EGFR)] and PD98059 (a specific inhibitor of the activation of mitogen-activated protein kinase) were used to help to determine the PTP1B signaling mechanism. Western blot analysis verified expression of EGFR and extracellular signal-regulated kinase (ERK) in ARPE-19 cells. The effect of siRNA-PTP1B on cell differentiation was confirmed by immunostaining for α-smooth muscle actin (α-SMA) and qRT-PCR. Cell migration ability was analyzed by transwell chamber assay.RESULTSThe mRNA levels of PTP1B were reduced by siRNA-PTP1B as determined by qRT-PCR assay. SiRNA-PTP1B activated EGFR and ERK phosphorylation. α-SMA staining and qRT-PCR assay demonstrated that siRNA-PTP1B induced retinal pigment epithelium (RPE) cells to differentiate toward better contractility and motility. Transwell chamber assay proved that PTP1B inhibition improved migration activity of RPE cells. Treatment with AG1478 and PD98059 abolished siRNA-PTP1B-induced activation of EGFR and ERK, α-SMA expression and cell migration.CONCLUSIONPTP1B inhibition promoted myofibroblast differentiation and migration of ARPE-19 cells, and EGFR/ERK signaling pathway played important role in migration process.

Purification and Characterization of Protein Tyrosine Phosphatase MEG1 and Preparation of Anti-PTPMEG1 Antibody

PTPMEGI is an intracellular protein tyrosine phosphatase（PTP）, which contains FERM and PDZ domains This study focuses our attention on the expression, purification and characterization of catalytic domain of PTPMEG1 （AMEG1） and preparation of its polyclonal antibody. A cDNA fragment encoding AMEG1 protein（amino acid residues 643-926） was amplified by PCR and then cloned into the pT7-7 vector. Both soluble and insoluble recombinant AMEG1 proteins were observed after induction by IPTG. Soluble AMEG1 was purified via two chromatographic steps, and the purified enzyme was characterized. With para-nitrophenylphosphate（pNPP） as a substrate, AMEG1 exhibited typical enzymatic characteristics of classic PTPs and classical Michaelis-Menten kinetics. Insoluble AMEG1, which was mainly distributed in the inclusion body of E. coli cells extracts, was purified by preparative electrophoresis gel for the preparation of the polyclonal antibody. A rabbit was immunized with AMEG1 purified by preparative electrophoresis to generate anti-AMEG1 antibody. Anti-serum was collected on 28th day after initial injection and purified via affinity chromatography. The purified polyconal antibody displayed a satisfactory titer and sensitivity.

POM analysis and computational interactions of 8-hydroxydiospyrin inside active site of protein tyrosine phosphatase 1B

Proteintyrosine phosphatase 1B(PTP1B)inhibitionis consideredas a potentialtherapeuticfor the treatmentof cancer,type2 diabetes,andobesity.Inour presentwork,weinvestigatedtheanti-diabeticpotentialof8-hydroxydiospyrin(8-HDN)from D.lotus against the PTP1B enzyme.It showed significant inhibitory activity of PTP1B with an IC 50 value of 18.37±0.02μM.A detailed molecular docking study was carried out to analyze the binding orientation,binding energy,and mechanism of inhibition.A comparative investigation of 8-HDN in the catalytic,as well as the allosteric site of PTP1B,was performed.Binding energy data showed that compound 8-HDN is more selective for the allosteric site and hence avoids the problems associated with catalytic site inhibition.The inhibition mechanism of 8-HDN can be further investigated as an active lead compound against PTP1B by using in vitro and in vivo models.

Structural Insight into the Design on Oleanolic Acid Derivatives as Potent Protein Tyrosine Phosphatase 1B Inhibitors

Oleanolic acid derivatives act as newer protein tyrosine phosphatase 1B （PTP-1B） inhibitors for type 2 diabetes mellitus （T2DM）. In order to understand the structural requirement of PTP-1B inhibitors, 52 oleanolic acid derivatives were divided into a training set （34 compounds） and a test set （18 compounds）. The highly reliable and predictive 3D-QSAR models were constructed by CoMFA, CoMSIA and topomer CoMFA methods, respectively. The results showed that the cross validated coefficient （q2） and non-cross-validated coefficient （R2） were 0.554 and 0.999 in the CoMFA model, 0.675 and 0.971 in the CoMSIA model, and 0.628 and 0.939 in the topomer CoMFA model, which suggests that three models are robust and have good exterior predictive capabilities. Furthermore, ten novel inhibitors with much higher inhibitory potency were designed. Our design strategy was that （i） the electronegative substituents （Cl, -CH2OH, OH and -CH2Cl） were introduced into the double bond of ring C, （ii） the hydrogen bond acceptor groups （C≡N and N atom）, electronegative groups （C≡N, N atom, -COOH and -COOCH3） and bulky substituents （C6H5N） were connected to the C-3 position, which would result in generating potent and selective PTP-1B inhibitors. We expect that the results in this paper have the potential to facilitate the process of design and to develop new potent PTP-1B inhibitors.

Synthesis and protein tyrosine phosphatase 1B inhibition activities of two new synthetic bromophenols and their methoxy derivatives

3-bromo-4,5-bis（2,3-dibromo-4,5-dihydroxybenzyl）-l,2-benzenediol （1） is a natural bromophenol isolated from the red algae Rhodomela confervoides that exhibits significant inhibition against protein tyrosine phosphatase 1B （PTP1B）. Based on its activity, we synthesized two new synthetic bromophenols and their methoxy derivatives from vanillin using the structure of natural bromophenol 1 as a scaffold. The structures of these bromophenols were elucidated from H NMR, 13C NMR, and high resolution electron ionization mass spectrometry as 2,3-dibromo-1-（2＇-bromo-6＇-（3＂,4＂-dimethoxybenzyl）- 3 ＇,4 ＇-dimethoxybenzyl）-4,5 -dimethoxybenzene （2）, 2,3-dibromo- 1 -（2 ＇-bromo-6＇-（2 ＂-bromo-4＂,5 ＂-dimethoxy- benzyl）-3＇,4＇-dimethoxybenzyl）-4,5-dimethoxybenzene （3）, 3,4-dibromo-5-（2＇-bromo-6＇-（2＂-bromo-4＂,5＂- dihydroxybenzyl）-3＇,4＇-dihydroxybenzyl）pyrocatechol （4） and 3,4-dibromo-5-（2＇-bromo-6＇-（3＂,4＂- dihydroxybenzyl）-3＇,4＇-dihydroxybenzyl）pyrocatechol （5）. PTP1B inhibition activities of these compounds were evaluated using a colorimetric assay, and compounds 3 and 4 demonstrated interesting activity against PTP1B.

A novel protein tyrosine phosphatase 1B inhibitor with therapeutic potential for insulin resistance

Insulin sensitizing medicines are currently limited, and identification of new drug candidate is a chal- lenge. Protein tyrosine phosphatase 1B （PTP1 B） negatively regulates insulin signaling pathway, and its inhibition is anticipated to improve insulin resistance. This study investigated the pharmacological profiles of compound CX08005, a new PTP1B inhibitor, with therapeutic potential for insulin resistance in vivo and in vitro, respective- ly. Recombinant human PTP1B protein was used to measure the enzyme activity. The docking simulation was per- formed to explore the interactions between the compound and the protein. The insulin sensitivity was evaluated in Diet-induced obesity mice and/or T2DM KKAy mice by glucose tolerance test （GTT）, the blood glucose level, glucose stimulated insulin secretion （GSIS）, homeostasis model assessment of insulin resistance index （HOMA-IR） and the whole-body insulin sensitivity （ISwb） index, respectively. The hyperinsulinemic-euglycemic clamp was performed to evaluate the insulin stimulated glucose disposal both in whole body and in insulin-sensitive tissues （muscle and fat）. Furthermore, its direct effect in muscle, fat and liver cells was observed. We found that CX08005 was a competitive inhibitor of PTP1B with dose-dependent activity （IC50=5.95 × 10^-7 M）. Docking simulation demonstrated that CX08005 binds to PTP1B at the catalytic P-loop through hydrogen bonds. In DIO mice, treatment with CX08005 effectively ameliorated glucose intolerance in a dose-dependent manner （50- 200 mg. kg^-1 · d^-l）, and decreased HOMA-IR values. We also demonstrated that oral administration of 50 mg ~ kg^-1· d^-1 CX08005 improved hyperglycemia, hyperinsulinemia, HOMA-IR and ISwb in KKAy mice. In hyperin- sulinemic-euglycemic clamp test, CX08005 increased glucose infusion rate and glucose uptake in muscle and fat of DIO mice. In 3T3-L1 adipocytes and C2C12 myotubes, CX08005 enhanced insulin-induced glucose uptake. In HepG2 hepatocyte, CX08005 enhanced insulin-stimulated tyrosine phosphorylation of IRβ/IRS1 in a dose-depend- ent manner, respectively; furthermore, the phosphorylation of several downstream molecules, including Akt, Foxol and GSK3β was also increased, indicating this compound could augment insulin＇s ability to suppress hepatic glu- cose output （HGO）. Our results strongly suggest that compound CX08005 directly enhances insulin action in vitro and in vivo with therapeutic potential for insulin resistance.

Inactivation of Protein Tyrosine Phosphatase 1B （PTP1B） Activity by the Aqueous Partition of Guava Leaf Extract

Guava leaf tea has been used as a folk medicine for treating hyperglycemic conditions in Asia and Africa. The hypoglycemic efficacy of guava leaf has been documented by many scientists in these regions, but the hypoglycemic mechanism is poorly understood. Guava leaves were extracted with methanol and the crude extract was partitioned against hexane, ethyl acetate, and butanol in sequence. The leftover in water is defined as the aqueous partition. A second smaller batch was extracted with hot water directly. Oral glucose tolerance test was carried out on healthy mice instead of diabetic mice that lack endogenous insulin. Glucose uptake was examined with 3T3-L1 adipocytes. Oxidative effect on PTP1B （protein tyrosine phosphatase 1b） was carried out with real-time PTP1B enzymatic assay. The aqueous partition of guava leaf extract possesses a potent inhibitory effect on PTP1B enzymatic activity and this PTP1B inhibition is through a slow oxidative but reversible inactivation on the enzyme. The reversible inactivation would suggest guava leaf extract may augment PTP1B inhibition alongside the endogenous H2O2 which itself is induced by insulin. In addition, our study confirmed the hypoglycemic efficacy being associated with guava leaf and found the most effective molecules reside in the aqueous partition which is also less cytotoxic to Chinese hamster ovary cells when compared to other less polar partitions. The guava leaf extract can modulate insulin activity through a redox regulation on PP1B enzymatic activity. It is speculated that a compound similar to gallocatechin in the aqueous partition can reduce an oxygen molecule to hydrogen peroxide which in turn oxidizes the catalytic residue Cys in PTP1B. Therefore, the guava leaf tea can serve as a functional hypoglycemic drink that is suitable for either healthy or diabetic subjects.

胃癌组织中lncRNA PTPRG-AS1、miR-599表达与临床病理特征及预后的关系

目的分析胃癌组织中长链非编码RNA(long non-coding RNA,lncRNA)蛋白酪氨酸磷酸酶受体G基因反义链1(protein tyrosine phosphatase receptor G gene antisense chain 1,PTPRG-AS1)和miR-599表达水平,探讨其与临床病理特征及预后的关系。方法选取华中科技大学同济医学院附属梨园医院2016年1月至2020年2月收治的106例胃癌患者为研究对象。检测胃癌组织及瘤旁组织中lncRNA PTPRG-AS1和miR-599水平。结果胃癌组织中lncRNA PTPRG-AS1水平显著高于瘤旁组织,miR-599水平显著低于瘤旁组织(P<0.05)。lncRNA PTPRG-AS1与miR-599水平呈负相关(r=-0.485,P<0.05)。lncRNA PTPRG-AS1、miR-599均与TNM分期、浸润深度、分化程度、淋巴结转移相关(P<0.05)。lncRNA PTPRG-AS1低表达组、miR-599高表达组生存率显著升高(χ^(2)=8.206、6.881,P<0.05)。lncRNA PTPRG-AS1、miR-599表达是影响胃癌患者不良预后的危险因素(P<0.05)。结论胃癌组织中lncRNA PTPRG-AS1和miR-599表达水平与患者临床病理特征及预后密切相关。

Pachymic acid exerts antitumor activities by modulating the Wnt/β-catenin signaling pathway via targeting PTP1B

Objective:To determine the inhibitory effects of pachymic acid on lung adenocarcinoma(LUAD)cells and elucidate its underlying mechanism.Methods:CCK-8,wound healing,Transwell,Western blot,tube formation,and immunofluorescence assays were carried out to measure the effects of various concentrations of pachymic acid on LUAD cell proliferation,metastasis,angiogenesis as well as autophagy.Subsequently,molecular docking technology was used to detect the potential targeted binding association between pachymic acid and protein tyrosine phosphatase 1B(PTP1B).Moreover,PTP1B was overexpressed in A549 cells to detect the specific mechanisms of pachymic acid.Results:Pachymic acid suppressed LUAD cell viability,metastasis as well as angiogenesis while inducing cell autophagy.It also targeted PTP1B and lowered PTP1B expression.However,PTP1B overexpression reversed the effects of pachymic acid on metastasis,angiogenesis,and autophagy as well as the expression of Wnt3a andβ-catenin in LUAD cells.Conclusions:Pachymic acid inhibits metastasis and angiogenesis,and promotes autophagy in LUAD cells by modulating the Wnt/β-catenin signaling pathway via targeting PTP1B.

miR-362-3p通过MAPK1/PTEN/AKT信号通路调节冠心病大鼠心肌细胞凋亡和内皮细胞损伤

目的:通过构建冠心病大鼠模型,探讨微小RNA-362-3p(miR-362-3p)是否通过调节有丝分裂原活性蛋白激酶1(MAPK1)、蛋白酪氨酸磷酸酶基因(PTEN)、蛋白激酶B(AKT),参与影响冠心病大鼠的心肌细胞凋亡和内皮细胞损伤。方法:构建冠心病大鼠模型,随即将大鼠分为健康组、冠心病组、miR-362过表达组、过表达阴性对照组、过表达+MAPK1激活组,苏木精-伊红(HE)染色观察各组大鼠冠状动脉组织病理学变化;原位末端标记测定法(TUNEL)检测各组大鼠心肌细胞凋亡情况;酶联免疫吸附法(ELISA)检测血清中炎性相关因子及一氧化氮(NO)、内皮素(ET)-1等含量;蛋白免疫印迹法(Western Blot)检测各组大鼠冠状动脉组织中MAPK、磷酸化MAPK(p-MAPK)、PTEN、AKT、磷酸化AKT(p-AKT)信号通路蛋白表达水平。结果:健康组大鼠冠状动脉组织完好;与健康组比较,冠心病组大鼠冠状动脉组织增厚明显,心肌细胞凋亡率、血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、ET-1含量及冠状动脉组织p-MAPK/MAPK、PTEN蛋白表达量升高,NO含量、p-AKT/AKT降低(P<0.05);与过表达阴性对照组比较,miR-362过表达组冠状动脉组织厚度减小,心肌细胞凋亡率及血清TNF-α、IL-1β、IL-6、ET-1含量、冠状动脉组织p-MAPK/MAPK、PTEN蛋白表达量降低,NO含量、p-AKT/AKT升高(P<0.05);与miR-362过表达组比较,过表达+MAPK1激活组冠状动脉组织增厚明显,且心肌细胞凋亡率、血清TNF-α、IL-1β、IL-6、ET-1含量及冠状动脉组织p-MAPK/MAPK、PTEN蛋白表达量升高,NO含量、p-AKT/AKT降低(P<0.05);冠心病组与过表达阴性对照组比较,各项指标比较差异无统计学意义(P>0.05)。结论:miR-362-3p过表达可调控MAPK1/PTEN/AKT信号通路,缓解内皮细胞损伤,减轻心肌组织损伤,从而改善冠心病病情。

初发1型糖尿病患儿血清GADA抗体和IA-2A抗体的阳性率及其与糖脂代谢紊乱的关系

目的探讨初发1型糖尿病(T1DM)患儿血清谷氨酸脱羧酶抗体(GADA)和蛋白酪氨酸磷酸酶抗体(IA-2A)阳性率及其与糖脂代谢紊乱的相关性。方法选取2018年1月-2022年1月在南京市高淳人民医院内分泌科住院治疗的初发1型糖尿病患儿95例为T1DM组,另选取同期体检的95例健康儿童作为对照组,对比两组GADA抗体和IA-2A抗体阳性率及糖脂代谢指标的水平,同时分析T1DM组中GADA抗体和IA-2A抗体阳性率与糖脂代谢指标的相关性。结果T1DM组血清糖化血红蛋白(HbA1c)、空腹血糖(FPG)、餐后2 h血糖(2 h-PG)、总胆固醇(TC)、甘油三酯(TG)及低密度脂蛋白胆固醇(LDL-C)水平高于对照组,血清高密度脂蛋白胆固醇(HDL-C)水平低于对照组,差异具有统计学意义(P<0.05)。对照组血清GADA抗体阳性率为2.11%,IA-2A阳性率为1.05%;T1DM组血清GADA抗体阳性率为46.32%,IA-2A阳性率为22.11%;T1DM组2种抗体的阳性率显著高于对照组,差异具有统计学意义(P均<0.05)。T1DM组中血清GADA抗体阳性者和IA-2A抗体阳性者有较高的HbA1c、FPG、2 h-PG、TC、TG及LDL-C水平,同时有较低的血清HDL-C水平,差异具有统计学意义(P<0.05)。T1DM组患儿血清GADA和IA-2A抗体水平与HbA1c、FPG、2 h-PG、TC、TG及LDL-C水平呈显著正相关(P<0.05),与HDL-C水平呈显著负相关(P<0.05)。结论T1DM患儿血清GADA抗体及IA-2A抗体有较高的阳性率,同时与糖脂代谢呈显著的相关性,T1DM患儿的发病及糖脂代谢紊乱可能与GADA和IA-2A抗体的阳性率升高有关。

Bioactive chemical constituents from the marine-derived fungus Cladosporium sp.DLT-5

A new isochromanone,cladosporinisochromanone(1),accompanied by 15 known compounds(2–16)were obtained from secondary metabolites produced by marine-derived fungus Cladosporium sp.DLT-5.NMR and HRESIMS spectra elucidation determined the planar structure of 1.Subsequent electronic circular dichroism(ECD)experiment assigned the absolute configuration of 1.Compounds 1,2,4–6,and 10 displayed different degrees of neuroprotective activities on human neuroblastoma cells SH-SY5Y.Five compounds(1,3–5,and 13)emerged resistance to protein tyrosine phosphatase 1B(PTP1B),further kinetic analysis and molecular docking study indicated that the most potent compound 13(IC50value of 10.74±0.61μmol/L)was found as a noncompetitive inhibitor for PTP1B.Surface plasmon resonance(SPR)and molecular docking studies also demonstrated the interaction between compound 12 and Niemann-Pick C1 Like 1(NPC1L1),which has been identified as significant therapeutic target for hypercholesteremia.In addition,compounds 3,6,and 14 showed attractive inhibitory activity against the phytopathogenic fungi:Colletotrichum capsici.Therefore,library of Cladosporium metabolites is enriched and new active uses of known compounds are explored.

外周血PTPN3、DCLK1水平与卵巢癌患者临床病理特征及预后的关系

目的探究外周血蛋白酪氨酸去磷酸酶3(PTPN3)、双皮质素样激酶1(DCLK1)水平与卵巢癌(OC)患者临床病理特征及预后的关系。方法选取2018年5月至2019年5月在张家口市第一医院收治的120例疑似OC患者作为研究对象,将60例经手术病理诊断确诊为OC的患者纳入OC组,60例经手术病理诊断确诊为良性的患者纳入良性组;选取同期同一医院体检的60例健康女性纳入对照组。根据OC患者3年随访情况,分为生存组(n=41)和死亡组(n=19)。采取酶联免疫吸附试验(ELISA)法检测所有研究对象外周血PTPN3、DCLK1水平。采用Kaplan-Meier法分析PTPN3、DCLK1水平与患者预后的关系,COX回归分析影响OC患者预后的危险因素。结果与对照组相比,OC组和良性组外周血PTPN3、DCLK1水平显著升高;与良性组相比,OC组外周血PTPN3、DCLK1水平显著升高(P<0.05)。PTPN3和DCLK1水平与OC患者国际妇产科联盟(FIGO)分期、有无淋巴结转移、分化程度有关(P<0.05)。PTPN3、DCLK1高表达组患者3年内生存率低于低表达组(P<0.05)。死亡组外周血PTPN3、DCLK1水平显著高于生存组(P<0.05)。多因素COX回归分析结果显示,分化程度、高水平PTPN3及DCLK1是影响OC患者预后的危险因素(P<0.05)。结论OC患者外周血PTPN3、DCLK1表达水平显著升高,且与OC患者术后3年的生存状况相关。

Protein tyrosine phosphatase 1B inhibitory activities of ursane-type triterpenes from Chinese raspberry, fruits of Rubus chingii

Protein tyrosine phosphatase 1 B(PTP1 B) has led to an intense interest in developing its inhibitors as anti-diabetes, anti-obesity and anti-cancer agents. The fruits of Rubus chingii(Chinese raspberry) were used as a kind of dietary traditional Chinese medicine. The methanolic extract of R. chingii fruits exhibited significant PTP1 B inhibitory activity. Further bioactivity-guided fractionation resulted in the isolation of three PTP1 B inhibitory ursane-type triterpenes: ursolic acid(1), 2-oxopomolic acid(2), and 2α, 19α-dihydroxy-3-oxo-urs-12-en-28-oic acid(3). Kinetics analyses revealed that 1 was a non-competitive PTP1 B inhibitor, and 2 and 3 were mixed type PTP1 B inhibitors. Compounds 1-3 and structurally related triterpenes(4-8) were further analyzed the structure-activity relationship, and were evaluated the inhibitory selectivity against four homologous protein tyrosine phosphatases(TCPTP, VHR, SHP-1 and SHP-2). Molecular docking simulations were also carried out, and the result indicated that 1, 3-acetoxyurs-12-ene-28-oic acid(5), and pomolic acid-3β-acetate(6) bound at the allosteric site including α3, α6, and α7 helix of PTP1 B.

Change of glutamic acid decarboxylase antibody and protein tyrosine phosphatase antibody in Chinese patients with acute-onset type 1 diabetes mellitus

Background Glutamic acid decarboxylase antibody （GADA） and protein tyrosine phosphatase antibody （IA-2A） are two major autoantibodies, which exert important roles in the process of type 1 diabetes mellitus （T1D）. Our study aimed to investigate the changes in positivity and titers of GADA and IA-2A during the course of Chinese acute-onset T1D patients and their relationships with clinical features.

1型糖尿病一级亲属GADA、IA-2A与IAA联合检测的价值

目的 采用放射配体法检测118 例1 型糖尿病一级亲属的谷氨酸脱羧酶抗体(GA-DA)、蛋白酪氨酸磷酸酶抗体(IA-2A)、胰岛素自身抗体(IAA),发现三种抗体单独检出率与欧美高加索人接近,联合检测阳性率优于单一抗体检测。

SHP-1在食管鳞状细胞癌组织中异常低表达的表观遗传学调节机制及其临床意义

目的:探讨食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)组织中蛋白酪氨酸磷酸酶1(protein tyrosin phosphatase 1,SHP-1)基因异常低表达的表观遗传学调节机制及其临床意义。方法:所用组织标本均来自河北医科大学第四医院2008-2011年行食管癌根治术并且病理诊断为ESCC患者的癌组织及相应癌旁组织(距癌灶边缘2 cm以上),共71例。ESCC细胞株(Eca109、Kyse170、Yes-2)培养完成后,进行甲基化抑制剂5-Aza-dC或组蛋白去乙酰化酶抑制剂TSA处理,qPCR和Western blotting实验检测ESCC组织和细胞系中SHP-1 mRNA和蛋白的表达变化,亚硫酸氢盐基因组测序(bisulfite genome sequencing,BGS)法检测ESCC细胞系中SHP-1基因启动子区CpG位点的甲基化频率,甲基化特异性PCR(methylation specific PCR,MSP)技术检测ESCC组织和细胞中SHP-1启动子区的甲基化状态,应用双荧光素酶报告基因实验检测SHP-1启动子区CpG岛甲基化对其转录活性影响。分析ESCC组织中SHP-1甲基化状态分别与临床病理特征和SHP-1 mRNA表达的关系,对组织SHP-1甲基化水平与ESCC患者生存率进行Kaplan-Meier生存分析和Log-Rank检验。结果:5-Aza-dC处理后,SHP-1 mRNA蛋白在3种细胞株中的表达显著上调(均P<0.05),同时其启动子区的甲基化程度均明显降低(均P<0.05);应用TSA处理细胞株后,SHP-1在各细胞株中的表达情况及甲基化状态无明显改变(P>0.05);甲基转移酶处理细胞荧光素报告载体活性显著低于未处理细胞荧光素报告载体活性(P<0.05),表明SHP-1的甲基化可抑制自身的转录。ESCC组织中启动子区的甲基化率明显高于癌旁组织(P<0.05),并与TNM分期、病理分级及淋巴结转移密切有关(P<0.05);与癌旁组织相比,ESCC组织中SHP-1 mRNA相对表达量显著降低(P<0.05),并与启动子区甲基化有关(P<0.05);Kaplan-Meier分析显示,启动子区高甲基化与ESCC患者的不良预后有关(P<0.05)。结论:ESCC组织和细胞株中SHP-1基因启动子区高甲基化状态可抑制其自身的转录活性,进而导致该基因表达沉默;SHP-1的高甲基化与ESCC患者预后不良有关,SHP-1的甲基化状态可能成为ESCC患者预后的评估指标。

人IA-2基因的部分区段克隆表达及其在1型糖尿病诊断中的应用价值评估

目的:构建人IA-2基因不同区段原核表达载体,诱导表达获得重组蛋白,并初步验证其在1型糖尿病蛋白酪氨酸磷酸酶自身抗体检测中的价值。方法:用RT-PCR方法调取目的基因,构建相应的原核表达质粒,转化大肠杆菌HB101,诱导表达获得纯化重组蛋白;以重组蛋白为包被抗原,初步建立检测蛋白酪氨酸磷酸酶自身抗体的ELISA方法,评价各片段在1型糖尿病诊断中的价值。结果:获得了2种可被1型糖尿病患者血清识别的重组人蛋白酪氨酸磷酸酶抗原区段IA-2(601～979)和IA-2(683～979),检测敏感性和特异性相当,但IA-2(683～979)检测的阳性D450nm值明显高于IA-2(601～979),成为首选的抗原区段。结论:所选重组人IA-2(683～979)抗原区段具有良好的抗原性,可作为1型糖尿病患者辅助诊断试剂的候选抗原。

Inhibition of protein tyrosine phosphatase 1B activity by triterpenes isolated from Aceriphyllum rossii

An organic layer prepared from the seed of Aceriphyllum rossii was studied to identify the active compounds for protein tyrosine phosphatase 1B（PTP1B） inhibition.Bioassay guided fractionation resulted in the isolation of PTP1B inhibitory activity of triterpenes（1-4）.These four compounds were identified as aceriphyllic acid C（1）,aceriphyllic acid D（2）,aceriphyllic acid E（3） and aceriphyllic acid F（4）.The isolated 1-4 compounds inhibited PTP1B with IC50 values ranged from（2.1±1.5） μmol/L to（11.2±2.5） μmol/L.Kinetic analysis of PTP1B inhibition by aceriphyllic acid C（1） and aceriphyllic acid D（2） suggested that oleanane-type triterpenes inhibited PTP1B activity in a mixed-type manner.