Objective To observe the effect of Jianpi Yishen Huatan Huoxue Decoction on type 2 diabetes insulin resistance Disease.Methods60 patients were randomly divided into treatment group and control group. Both groups were treated by Mefformin, 0.5 tid. On this basis, Patients in the treatment group were treated by Jianpi Yishen Huatan Huoxue Decoction, with one preparation daily for twice uses. The therapeutic course for both groups was eight weeks.ResultsIn the treatment group, the distinct effective rate and the total effective rate were 50% and 83% respectively for diabetes type 2insulin resistance Disease on TCM Syndrome. In the control group, the distinct effective rate and the total effective rate were 30% and 63% respectively. The improvement in the treatment group was significantly higher than the control group, in terms of insulin sensitivity index of FINS and IAI and TG and HDL-C in lipid.ConclusionJianpi Yishen Huatan Huoxue Decoction is effective to the patients of type 2 diabetes insulin resistance Disease.

Objective To detect the proteins structure encoded by COL4A4 gene with different missense mutations of thin basement membrane nephropathy (TBMN) and to analyze the effect of gene mutation on the secondary structure of α4 (Ⅳ) chain and its association with phenotype. Methods A COL4A4-linked TBMN patient with FSGS by a missense mutation (g. 1214G>A resulting in p. G405E) diagnosed by clinical manifestations, family history and renal biopsy examination, as well as two controls (one healthy, one pure TBMN carrying a g. 1550G>A mutation resulting in p. G448S) were enrolled in this study. The fragments of cDNA with the two mutations and that of corresponding cDNA from the healthy control were expressed in E. coll. The secondary structures of recombinant polypeptides were analyzed by circular dichroism (CD) spectroscopy. Results CD spectra of healthy control exhibited a negative peak near 208 nm whereas that of TBMN patient with FSGS exhibited a negative peak near 220 nm. Furthermore, the magnitude of the negative peak of this patient decreased as compared with that of healthy control. CD spectra of pure TBMN control was slightly changed with the negative peak remaining near 208 run and the magnitude slightly decreased as compared with that of healthy control. In addition, the secondary structure of pelypeptide from healthy control was composed of about 1/4 α-helix and 1/4 β-sheet, whereas that from the patient presented about 1/3 α-helix without any β-sheet. The secondary structure of polypeptide from pure TBMN control was almost the same as the healthy control, except a shght reduction of α-helix and a slight increase of β-sheet. Conclusions Although the glycine substitutions exists in the nearby domain of α4 (Ⅳ)chain, the TBMN patient complicating FSGS with severe phenotype and g. 1214G>A mutation and the pure TBMN control with the mild phenotype and g. 1550G>A mutation are revealed with different secondary structures of α4 (Ⅳ)chain. Moreover, the secondary structure change of α4 (Ⅳ) chain is consistent with their corresponding phenotype severity.

Objective Studies show that the abnormal expression of EphB4 plays an important role in the development and progression of colon cancer .The present study aims to provide some experimental evidence for the gene therapy of colon cancer by con -structing a lentiviral expression vector carrying the homo EphB4 gene and further establishing colon cancer cell lines with stable overex-pression of EphB4. Methods A series of oligonucleotides (oligo) encoding the homo EphB4 gene were ligated together by PCR and then cloned into a lentiviral expression vector pLenti 6.3-MCS-IRES2-EGFP.After confirmed by sequencing , the vector pLenti6.3-EphB4-IRES2-EGFP and its helper vectors were mixed and co-transfected into 293 T cells to obtain recombinant virus containing the EphB4 gene.The lentiviral titer was detected and the resulting recombinant lentiviruses carrying EphB4 or control viruses only carrying green fluorescence protein (GFP) were used to infect the human colon cancer cell lines .The expression of GFP was determined under the inverted fluorescence microscope and the level of EphB 4 mRNA in the infected cells detected by qPCR . Results The lentiviral expression vector pLenti6.3-EphB4-IRES2-EGFP carrying correct homo EphB4 gene sequence was successfully constructed .The titer of the recombinant EphB4 lentiviral supernatant Lenti6.3-EphB4 was 1 ×108 TU/mL.The expression of GFP was observed in the trans-duced cells under the fluorescence microscope , and that of EphB4 mRNA in the transfected SW480 and Coca-2 cells was significantly up-regulated as compared with the control and blank groups . Conclusion The homo EphB4 gene was successfully amplified and cloned.A lentiviral expression vector was successfully constructed , and so were colon cancer cell lines stably overexpressing EphB 4, which may shed light on the lentivirus-mediated genetic therapy for colon cancer .

Objective Studies show that the role of EphB 4 in the development and progression of cancer is correlated to its ligand EphrinB2.The present study was to observe the effect of the changes in EphB4 on the expression of EphrinB2 by constructing and identifying microRNA ( miRNA) interference vectors targeting the EphB4 gene in colon cancer cells . Mte hods According to the EphB4 gene sequence , 3 pairs of oligo DNA sequences of miRNA were designed .The single strand of oligo DNA was annealed to form double-strand DNA, and then connected with the plasmid pcDNA 6.2-GW/EmGFP-miRNA.The expression vector pcDNA6.2-GW/EmGFP-miR-EphB4 was linked to pDONR221 and pLenti6/V5-DEST to construct the lentiviral expression vector pLenti 6/V5-DEST-EphB4, which was cotransfected with packaging mix (pLP1, pLP2 and pLP/VSVG) into 293FT cells by lipofectamine 2000 transfec-tion to produce lentivirus , and the lentivirus titer was measured by infection of HEK 293 cells.The stable cell lines were selected and cultured.The expression levels of EphB4 and EphrinB2 were examined by qPCR. Results Three miRNA interference vectors SR-1, SR-2, and SR-3 targeting the EphB4 gene were successfully constructed , with SR-3 exhibiting the most significant interference efficien-cy.The constructed lentiviral vector pLenti 6/V5-DEST-EphB4 was successfully packaged in 293FT cells.The virus titer was 7 ×108 Caco-2 cells. Conclusion The exogenous EphB4 expression could be significantly inhibited by treatment with specific miRNA in co-lon cancer cells .The correlation of EphB4 and EphrinB2 may be effected by many factors and need further studies .

Objective To investigate the occupational stress and mental health of the staffs of disabled care agency, and the main factors related. Methods The staffs were investigated with Work Stress Scale, Coping Style Questionnaire, and Symptom Checklist (SCL-90). Results The work stress was hard in the subjects with poor mental health condition. Their cope style was mainly positive and mature. Gender,levels of education, jobs, and coping styles related with the work stress, while the work stress and coping style related with the mental health. Conclusion The staffs in disabled care agency experience work stress and erious mental problems, which need to be solved.

Purpose To explore the immunohistochemical ( IHC) expression of ALK antibodies with different clones in anaplastic lym-phoma kinase ( ALK) gene fusion non-small cell lung cancer ( NSCLC) . Methods ALK expression in 60 NSCLCs were detected by IHC including autostainer (D5F3, Ventana+BenchMark) and manual staining using 4 different antibodies of D5F3 (Ventana), D5F3 (Cell Signaling), 1A4/1H7 (OriGene), 5A4 (Abcam), and all cases were verified with ALK FISH. Their expressions with 4 anti-bodies were compared with those by D5F3 (Ventana+BenchMark). Results 32 ALK gene rearrangement NSCLCs and 28 negative cases were identified by FISH and D5F3 (Ventana+BenchMark). The sensitivity of D5F3 (Ventana), D5F3 (Cell Signaling), 1A4/1H7 (OriGene), 5A4 (Abcam) was 93. 8%, 84. 4%, 93. 8%, 56. 3%, and all the speciticity was 100%. The consistency with D5F3 (Ventana+BenchMark) was 96. 7%, 91. 7%, 96. 7% and 76. 7%, respectively. The validity of immunohistochemical staining in surgical resection specimens was better than in small biopsies. Conclusion Effective routine manual immunohistochemistry with high-affinity antibody clone may provide a more economic and widespread pre-screening technique.

Objective To elucidate the clinicopathological and hereditary characteristics in Fabry nephropathy.Methods The clinical and pathological features of 14 patients with Fabry nephropathy were collected.The activities of α-Gal A were measured in 4 probands.Screenings of GLA mutations were done in 12 patients.Results The ratio of Fabry nephropathy in the patients with renal biopsy was 0.074 ％ (14/19 005),the average diagnostic age was (30.57±9.32) years,male to female ratio was 2.5∶ 1.All the patients had proteinuria,and the median of urine total protein (UTP)was 1.71 g/24 h (0.32-4.71 g/24 h).Two of them got nephrotic proteinuria,5 of them got microscopic hematuria,4 of them got renal function insufficiency.Angiokeratomas was the most common manifestation (10/14),followed by cardiac involvement (6/14).Hypohidrosis and diseases of central neural system could also be seen in these patients.There were 9 classic phenotype,the remaining 5 were variants/renal variants.The family information was collected in 10 pedigrees,6 of them had family histories of kidney disease.Renal pathology showed vacuolization of glomerular visceral epithelial cells was the prominent feature,global and segmental glomerulosclerosis were seen in some patients by light microscopy.The myelin bodies or zebra bodies were identified in podocytes by electron microscopy,but only were found in some podocytes of 2 females.The activity of α-Gal A was decreased compared with the normal range in 4 probands.The mutations of GLA were demonstrated in 11 patients.Three novel mutations of GLA gene were identified,which were all deletion/insertion mutations with classic phenotypes.Most variants carried the mutations located in the buried/partial buried areas of α-Gal A (3/11).The classical phenotype carried GLA mutations as W162X,F169S,S201F,N272K,L310R,while variant phenotype carried GLA mutations as I91T,R112H,Q312H.Conclusions The ratio of Fabry nephropathy in patients with renal biopsy is 0.074％.Angiokeratomas and cardiac involvement are often accompanied with Fabry nephropathy.The genotypes of GLA may have close relationships with the phenotypes of Fabry nephopathy.

Objective To elucidate whether focal segmental glomerulosclerosis (FSGS) is a secondary development of the COL4-linked thin basement membrane nephropathy (TBMN) or the primary FSGS produces thin glomerular basement membrane (GBM). Methods The family members presented microscopic hematuria,increasing proteinuria with the years and a dual pathological diagnosis of FSGS and TBMN was made in the proband.DNA linkage analysis at locus 2q36-37 that contains the COL4A3/COL4A4 genes was performed with polymorphic micmsateilite markers D2S434,D2S279,D2S1370,D2S256 and D2S427.Haplotypes were constructed at the COL4A3/COL4A4 loci for affected and unaffected family members.All exons of COL4A3 and COL4A4 genes were screened for mutations in the proband.Mutation screening was also performed for NPHS1,NPHS2,CD2AP,WTI,TRPC6 and ACTN4 to exclude familial FSGS.Mutation or polymorphism found in the family were examined in 50 healthy controls. Results In this family hematuria segregated with the 55224 haplotype at the COL4A3/COL4A4 locus.G to A substitution at nucleotide 1214 resulting in an glycine being replaced by glutamate (G405E) was demonstrated for the first time in cxon 20 of COL4A4 gene.G4OSE was present in all four members of the family with hematuria but not in the seven unaffected family members nor in 50 healthy controls.A novel polymorphism segregating with hematuria (IVS1-4C＞T in exon 2 ofCOL4A3) was also found which was only present in all four affected family members but not in the seven unaffected family members. No mutations were demonstrated in FSGS associated genes,however,a novel SNP (R268Q),which distributed with the disease ineompletely,was described in the NPHS1 gene coding nephrin,the podocyte slit diaphragm protein. Conclusions In this family,FSGS occurres on the basis of TBMN.Maybe the particular COL4A3/COL4A4 mutation and polymorphism work together to develop proteinuria and eventually leading to FSGS.But whether the mutation and the polymorphism segregating with the disease predispose to develop FSGS in TBMN patients is required further study.

Objective To elucidate the features of clinicopathology and mutation in Fabry disease complicated with thin basement membrane nephropathy (TBMN), and to investigate the kindred. Methods Data of clinicopathology and gene mutation of a female patient of Fabry disease complicated with TBMN admitted to the Department of Nephro]ogy in our hospital were analyzed. Members of her kindred were investigated simultaneously. Results Proband was a 41-year-old Chinese woman who presented syndrome of Fabry disease and TBMN including angiokeratomas, chronic pain, tinnitus, vertigo, left ventricular hypertrophy and nephropathy as proteinuria, microscopic hematuria and hypertension. A percutaneous renal biopsy was performed on the proband, which was consistent with FSGS and vaculization of podocytes by light microscopy.Electron microscopy showed concentric lamellated inclusions in some podocytes. Diffuse thinning of glomerular basement membrane (GBM) with a mean thickness of (216±31) nm was found. The diagnosis of TBMN with suspected Fabry disease was identified. Family screening showed that her daughter had microscopic hematuria, tinnitus and neuropathic pain. One of her sisters only had microscopic hematuria. The activity of α-galacsidase A (α-Gal A )enzyme in the proband and her daughter was 33 units and 75 units respectively (the normal range is 100 to 500 units). They all carried the novel GLA mutation 1208 ins 21 bp and COL4A3 SNP c: 3627G＞A(p:M1209I). While her sister who only had microscopic hematuria just carried the variant of COL4A3 gene-c:3627G ＞A (p:M1209I), and had the normal activity of α-Gal A with no mutation of GLA.Conclusion TBMN should be considered in the patients of Fabry disease with the condition of benign familial hematuria.

Objective: To study the clinicopathological features, immunohistochemical phenotype, molecular changes, differential diagnosis and prognosis of eosinophilic solid and cystic renal cell carcinoma (ESC RCC). Methods: A total of 15 cases were selected from 2005 to 2019 at Nanjing Jinling Hospital,Nanjing University School of Medicine for clinicopathological and immunohistochemical analysis, 10 of which were subject to cancer-associated mutation analysis using targeted next-generation sequencing (NGS) panel. A literature review was also performed. Results: The patients′ ages ranged from 15 to 68 years (mean, 33 years). The male-to-female ratio was 1.1∶1.0. During a mean follow-up of 22 months, none of the patients developed tumor recurrence, progression or metastasis. Histologically, the tumors typically demonstrated solid and cystic architectures and the neoplastic cells contained voluminous eosinophilic cytoplasm with prominent granular cytoplasmic stippling. Immunohistochemically, tumor cells in all cases were immunoreactive for CK20. Signal pathway related protein mTOR and S6 were positive in 14/15 and 6/15 cases, respectively. Cathepsin K, Melan A and HMB45 were at least focally positive in 12/15, 6/15 and 2/15 cases, respectively. CK7 and CD10 showed focal immunostain positivity in some cases, while TFE3, TFEB, CA9 and CD117 were negative in all cases. NGS demonstrated TSC1/TSC2 mutations in all tested cases (10/10). Conclusions ESC RCC is a rare tumor that tends to occur in young patients with an indolent behavior. Diagnosis can be established by its distinct clinical and histopathologic findings, immunohistochemical phenotype and molecular genetics. The tumor may be considered as a new subtype of RCC.