We explored the improved method to prepare decellularized kidney scaffold and provide experimental basis for kidney tissue engineering and renal pathology and toxicology in vitro research. We perfused rat kidneys with PBS (group control) and prepared the decellularized kidney scaffolds with sodium dodecyl sulfate (SDS) (group S), Triton X-100 combined with SDS (group TS), and Triton X-100 combined with SDS after repeated freezing and thawing (group FTS) in different flow velocity. Meanwhile we measured their fluid distributions and vascular resistance. We examined the degree of decellularization of acellular scaffolds by HE, DAPI staining and DNA quantification. We examined the retention of main composition and structural integrity of decellularized scaffolds by Masson, PAS and immunohistochemical staining. We also detected the ultrastructure, cytotoxicity and the level of growth factor of the scaffolds by scanning electron microscope, MTT and ELISA, respectively. The results showed that the time of decellularization in group FTS was less than that in group S and TS. The vascular resistance of scaffolds decellularized at 10 mL/min flow velocity was lower. The fluid distribution in groups S, TS and FTS was different from that in control group. No residual cell was detected by HE and DAPI staining. DNA content was less than 50 ng/mg. Masson, PAS and immunohistochemical staining results showed that there was extracellular collagen, polysaccharide, type I collagen, type IV collagen, fibronectin and laminin in the decellularized scaffolds, and the scanning electron microscope result showed the scaffolds had the honeycomb structure. The cytotoxicity level of decellularized scaffolds was between grade 0 to 1. The level of VEGF, EGF, IGF-1 and PDGF-BB in group FTS were significantly higher than those in group S and TS. In concluding, combining freeze-thawing with perfusion can produce more ideal and effective whole organ decellularized scaffold of rat kidney, and make a foundation for the study of kidney tissue engineering and in vitro pathology and toxicology of kidney.
Animals ; Collagen ; Extracellular Matrix ; Freezing ; Kidney ; Perfusion ; Rats ; Tissue Engineering ; Tissue Scaffolds

OBJECTIVE: To investigate the transcriptome profile of genital tubercles (GTs) in male SD rats and explore the mechanism of hypospadias induced by Di (2-ethylhexyl) phthalate (DEHP). METHODS: Forty time-pregnant SD rats were randomly divided into 4 equal groups, namely GD16 group and GD19 group (in which the male GTs were collected on gestation day[GD]16 and GD19 for RNA-seq, respectively), control group and DEHP exposure group (with administration of oil and 750 mg/kg DEHP by gavage from GD12 to GD19, respectively).In the control and DEHP exposure groups, the GTs were collected from the male fetuses on GD19.5, and scanning electron microscopy and HE staining were used to observe the morphological changes.The differentially expressed genes (DEGs) in the GTs were screened using lllumina HiSeq 2000 followed by GO and KEGG enrichment analyses to characterize the transcriptome profile.Immunofluorescence assay was performed to verify the DEGs (Mafb) identified by RNA-seq results.Immunofluorescence assay and Western blotting were used to examine the expression levels of Mafb in the penile tissue. RESULTS: A total of 1360 DEGs were detected in the GTs between GD16 group and GD19 group by RNA-seq.Among these genes, 797 were up-regulated and 563 were down-regulated.These DEGs were mainly enriched in the cell adhesion plaque signaling pathway, axon guidance signaling pathway, and extracellular matrix receptor signaling pathway.Compared with that in GD16 group, Mafb was significantly up-regulated in GD19 group, which was consistent with the sequencing results.Mafb and β-catenin were significantly down-regulated in DEHP-exposed group compared with the control group ( < 0.01). CONCLUSIONS Mafb expression increases progressively with the development of GTs in male SD rats.DEHP exposure causes significant down-regulation of Mafb and β-catenin, suggesting that β-catenin signaling pathway that affects Mafb is related to DEHP-induced hypospadias in SD rats.
Animals ; Diethylhexyl Phthalate ; Female ; Gene Expression Profiling ; Humans ; Hypospadias ; MafB Transcription Factor ; Male ; Oncogene Proteins ; Phthalic Acids ; Pregnancy ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: explore the expression of miR-155-5p in Wilms tumor and its effect in regulating the proliferation, migration and apoptosis of Wilms tumor cells. METHODS: Specimens of tumor tissues and paired adjacent tissues were obtained from 40 patients with Wilms tumor for detection of the expression levels of miR-155-5p using RT-qPCR. Wilms tumor cell line G401 was transfected with miR-155-5p mimics and miR-155-5p inhibitor to induce miR-155-5p over-expression and its inhibition, respectively, and the changes in the cell proliferation, migration and apoptosis were assessed using cell counting kit-8 (CCK-8), wound healing assay and fl ow cytometry. RESULTS: RT-qPCR showed that the expression of miR-155-5p decreased significantly in Wilms tumor tissues as compared with normal kidney tissues and was significantly associated with TNM stage ( < 0.05). In G401 cells, over-expression of miR-155-5p significantly inhibited the cell proliferation and migration and promoted cell apoptosis ( < 0.05), and down-regulation of miR-155-5p obviously enhanced the proliferation and migration and suppressed apoptosis of the cells ( < 0.05). CONCLUSIONS miR-155-5p is down-regulated in Wilms tumor and its expression level is correlated with TNM stage. miR-155-5p participates in the progression of Wilms tumor by inhibiting the proliferation and migration and promoting apoptosis of the tumor cells, and may serve as a novel biomarker for diagnosis, therapy and prognostic evaluation of Wilms tumor.
Apoptosis ; Cell Movement ; Cell Proliferation ; Humans ; Kidney Neoplasms ; genetics ; MicroRNAs ; genetics ; Neoplasm Invasiveness ; Wilms Tumor ; genetics

Objective To establish and assess the new method of APTT assay based on the combinations of Mg2+and Ca2+for lupus anticoagulants(LA)measurements.Methods This prospective study included 309 trisodium citrate anticoagulated plasma samples from 244 random patients and 65 patients with different autoimmune diseases(AID)to establish and assess the method of LA measurement, respectively.Final concentrations of 0,2.0, 4.0, 8.0,16.0 mmol/L Mg2+were added into 25 mmol/L Ca2+solution, and Actin reagent was used to measured plasma APTT of 94 patients.The applied concentration of Mg2+-Ca2+solution was confirmed through the special and significant alteration of APTT from LA-positive and -negative plasma observed in the presence of Mg 2+(test solution).Based on Actin reagent use,the test solution and 25 mmol/L Ca2+solution were applied to measure APTT of patients and normal individuals, respectively, and the ratio of Mg2+-Ca2+-APTT to Ca2+-APTT(Mg2+-Ca2+-APTT indices)and normalized Mg2+-Ca2+-APTT indices(NAR)were calculated, respectively.Mixed plasma NAR was measured,and CV%was calculated to evaluate the repeatability and stability of Mg 2+-Ca2+-APTT method.APTT of 150 patients was measured with the test solution and Actin reagent to calculate Mg 2+-Ca2+-APTT indices, and normalized LA ratio was determined with dRVVT method.The applicability of Mg2+-Ca2+-APTT assay was assessed through comparisons of the results from the two methods.Finally, NAR and NLR of 65 patients with AID(including 26 SLE patients)were measured with Mg2+-Ca2+-APTT assay and dRVVT method, respectively, and ROC curve was also used to assess the efficacy of the two methods for LA measurements.Results In all LA-negative plasma,APTT increased from 28.1 ±4.5 s to 61.2 ±7.9 s in normal APTT group,47.2 ±8.9 s to 97.5 ±10.3 s in increased APTT group,and 27.6 ± 5.1 s to 61.2 ±7.9 s in ACA-positive group when Mg2+increased from 0 to 8 mmol/L in Mg2+-Ca2+solution(F=34.12, 38.9 and 28.35,P<0.01).Following increased Mg2+concentration, APTT shortened from 0 to 4.0 mmol/L, but simultaneously prolonged from 4.0 to 16.0 mmol/L in LA-positive plasma with prolonged or normal APTT(F=31.55 and 39.51, P<0.01), and APTT was significantly higher in 8.0 mmol/L than that in 4.0 mmol/L(P<0.001).The test concentration of Mg 2+/Ca2+solution was 4.0 mmol/L.The within, inter-day CV% of NAR was 1.39%,2.30%, and 3.44%, respectively. According to the judging criteria of <0.966 and >1.034 of Mg2+/Ca2+indices, there was 141 patients with increased indices and NLR <1.20, and 9 patients with decreased ones and NLR≥1.20 in all 150 patients.The area under ROC curve of NAR and NLR for LA detection was 0.913(95%CI:0.848-0.978) and 0.892(95%CI:0.817-0.966), respectively, and the cut-off value was 0.87 and 1.13, respectively. The sensitivity and specificity of NAR(85% and 77%)was higher than that of NLR(81% and 74%), respectively.The accordant rate of positive,negative,and total results between NAR and NLR was 94.4%, 98.5%,and 98%,respectively.Conclusion The method of APTT assay based on Mg2+combining Ca2+for LA measurements is feasible,and can be used to detect plasma LA of patients.

OBJECTIVETo evaluate the usage of Mayo staging system in Chinese patients with primary light chain (LC) amyloidosis.

METHODClinical data, treatment and outcome of 162 primary LC amyloidosis patients with Mayo Clinic staging in Peking Union Medical College Hospital from January 2009 to June 2015 were retrospectively analyzed.

RESULTSThe median age of 162 patients with Mayo Clinic 2004 stage was 57 (20-81) y, of them 62.3% were male. The number of patients with stage I to III were 44 (27.2%), 69 (42.6%), and 49 (30.2%), respectively. The median overall survival was not reached, 23 months and 12 months in patients with Mayo Clinic 2004 stage I, II, and III, respectively (P<0.001). Among 128 patients with Mayo Clinic 2012 stage, 48 patients (37.5%), 32 patients (25.0%), 32 patients (25.0%) and 16 patients (12.5%) were staged as Mayo Clinic 2012 stage 1 to 4, and the median OS was not reached, not reached, 13 months and 3 months, respectively (P<0.001).

CONCLUSIONMayo Clinic staging systems had important prognostic value in patients with primary LC amyloidosis.

Adult ; Aged ; Aged, 80 and over ; Amyloidosis ; diagnosis ; Female ; Humans ; Immunoglobulin Light-chain Amyloidosis ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Young Adult

OBJECTIVETo observe the effect of electroacupuncture(EA) at Zhongwan(CV 12) on the energy metabolism along the conception vessel(CV) in volunteers with yang-deficiency constitution,and to explore the relationship of electroacupuncture regulation and body constitution.

METHODSEighteen volunteers with mild constitution and 18 volunteers with yang-deficiency constitution were collected out of 200 students of Fujian University of TCM by body constitution questionnaire. Skin microcirculatory blood perfusion units (MBPU) at Danzhong (CV 17), Xiawan(CV 10) and Qihai(CV 6) of CV were measured by a laser Doppler flowmetry in the normal condition and after EA stimulation at Zhongwan(CV 12) for 20 min.

RESULTS(1)Before treatment, (1)MBPU values at Danzhong(CV 17), Xiawan(CV 10) and Qihai(CV 6) in the yang-deficiency constitution group were lower than those in the mild constitution group,but there was no statistical significance (both P>0. 05) except Danzhong(CV 17) (P<0. 01). (Z)As for the three acupoints in the mild constitution group, MBPU level of Danzhong(CV 17) was higher than that of Xiawan(CV 10) without statistical significance(P->0. 05),and MBPU values of Danzhong(CV 17) and Xiawan(CV 10) were higher than that of Qihai(CV 6) (both P<0. 01). (3About the three acupoints in the yang-deficiency constitution group, MBPU result of Danzhong(CV 17) was lower than the value of Xiawan(CV 10), but higher compared with Qihai(CV 6)(P<0. 05, P<0. 01). MBPU of Xiawan(CV 10) was higher than Qihai (CV 6) as well(P<0. 01). (2) MBPU values of Danzhong(CV 17), Xiawan(CV 10) and Qihai(CV 6) were increased apparently compared with those before treatment after EA stimulation at Zhongwan(CV 12) for 20 min in the two groups(all P<0. 01). (3) The rise rates of MBPU level about Danzhong(CV 17) and Qihai(CV 6) in the yang-deficiency constitution group were higher than those in the mild constitution group without statistical significance after EA at Zhongwan(CV 12) for 20 min(both P>0. 05).

CONCLUSIONThe energy metabolism in CV of volunteers with yang-deficiency constitution is declined, especially Danzhong(CV 17). EA can rise energy metabolism in CV of mild or yang-deficiency constitution volunteers through regulating MBPU along meridian.

Acupuncture Points ; Adolescent ; Adult ; Electroacupuncture ; Energy Metabolism ; Female ; Humans ; Male ; Meridians ; Microcirculation ; Skin ; blood supply ; metabolism ; Volunteers ; Yang Deficiency ; metabolism ; physiopathology ; therapy ; Young Adult

Objective To establish the method of the SYBR Green Ⅰ real‐time fluorescent quantitative PCR for detecting the se‐rum miR‐203 expression level ,and to detect the serum miR‐203 expression levels in the patients with cervical cancer ,cervical benign diseases and healthy controls .Methods The miR‐203 ,U6 stem loop RT primers and the PCR amplification primers were designed for conducting fluorescence quantitative PCR ,with U6 as the internal relative quantification ,the serum miR‐203 levels were com‐pared among different cervical diseases .Results The established method could specifically detect the amplification signal of serum miR‐203 ,the melting curve was single and PCR products were specific .The serum miR‐203 level in the patients with cervical cancer was significantly higher than that in the patients with benign cervical diseases such as hysteromyoma and cervicitis ,the difference was statistically significant (P<0 .05) .Conclusion The SYBR Green Ⅰ real‐time fluorescent quantitative PCR is a quick ,simple detection method with high sensitivity and good specificity ,which may have a better application prospect in cervical cancer auxiliary diagnosis .

To investigate the effects of Xuefu Zhuyu Decoction, a compound Chinese herbal medicine, on the proliferation and extracellular matrix synthesis of cardiac fibroblasts (CFs) from neonatal rats induced by angiotensin II, and to explore the mechanism of Xuefu Zhuyu Decoction in improving myocardial fibrosis.

AIM: To investigate the expression level of transcriptional factor Elf-1 in mononuclear cells, CD4~+ and CD8~+ T cells from umbilical cord blood (UCB). METHODS: Real-time PCR with SYBR green I technique was used for detecting the Elf-1 expression in mononuclear cells, sorted CD4~+ and CD8~+ T cells from 12 cases of umbilical cord blood. The relative mRNA expression level of Elf-1 was analyzed by a formula of 2~(-△Ct)×100%. The expression level of β_2-microglobulin gene (β_2M) was used as an endogenous reference. The peripheral blood from 10 cases of healthy adults was served as control. RESULTS: Elf-1 mRNA expressed in all blood samples collected from both UCB and healthy adults. The expression level showed apparent diversity in different individuals. The relative mRNA expression of Elf-1 in both mononuclear cells (18.55%±2.48%) and CD8~+ T cells (3.52%±0.45%) from UCB were significantly higher than those from healthy adults (9.16%±1.92%, 2.02%±0.27%, respectively, P<0.01, P<0.05). CONCLUSION: The results of Elf-1 expression level from umbilical cord blood indicate that the over-expression of Elf-1 gene in mononuclear cells and CD8~+ T cells might be one of the features of T cell immune state in umbilical cord blood.

Objective This study aimed to explore issues about dysandia female during pregnancy,peripartum and contraception and to supply reference for their reproductive health improvement. Methods Purposive sampling strategy was used. With the aid of an interview guide, 12 dysaudia women who met inclu-sion and exclusion criteria were invited to be interviewed. Interviews were recorded, validated and then ana-lyzed. Results Four themes were generated, limited knowledge, passive information acquisition, total depen-dence on family and reluctant to talk about contraception. Total dependence on family was noticeable during pregnancy and peripartum. Though little was known about health protection related to pregnancy, peripartum and contraception, participants were passive at seeking for relevant information. Conclusions Deaf women may experience more threats to reproductive health in relation to pregnancy, peripartum and contraception that they require more guidance and assistance.