To establish single- and double-transfected transgenic cells stably expressing hMATE1, hMATE1 cDNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pcDNA3.1(+) plasmid by virtue of both HindIII and Kpn I restriction enzyme sites. Subsequently, the recombined pcDNA3.1(+)- hMATE1 plasmid was transfected into MDCK, MDCK-hOCT1 or MDCK-hOCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 µg · mL(-1), all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The mRNA content of hMATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed hMATE1 mRNA. The cellular accumulation of metformin in MDCK-hMATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 µmol · L(-1) cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good hMATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving hMATE1 alone or together with hOCT1/2 in vitro.
Animals ; Biological Transport ; Cimetidine ; pharmacology ; DNA, Complementary ; Dogs ; Drug Interactions ; Humans ; Madin Darby Canine Kidney Cells ; Metformin ; pharmacology ; Organic Cation Transport Proteins ; genetics ; metabolism ; Transfection

BACKGROUND AND OBJECTIVEAs chemotherapy is generally used in the clinical treatment of cancer, the problem of multidrug resistance (MDR) of tumors against the chemotherapeutic agents becomes more and more serious. It has been the major cause for the failure of the chemotherapy. We detected the expressions of beta-catenin and tumor drug resistance related proteins, MRP2, P-gp, and Bcl-2, in esophageal squamous cell carcinoma (ESCC) to explore their function and correlation in the occurrence and development of MDR in ESCC.

METHODSWe used the tissue microarray technique, immunohistochemistry, and image analysis methods to detect the expressions of MRP2, P-gp, beta-catenin, and Bcl-2 proteins and analyze their relationships with clinical data in a ESCC tissue microarray consisting of 582 specimens of ESCC, 294 specimens of normal mucosa, 92 specimens of basal cell hyperplasia, and 87 specimens of dysplasia adjacent to cancer tissue.

RESULTSThe integral optical density (IOD) of MRP2 and Bcl-2, which was 195.7 +/- 175.9 x 10(3)) and 90.5 +/- 112.5 x 10(3)), respectively, was significantly higher in ESCC than in normal mucosa, which was 104.8 +/- 86.1 x103) and 25.2 +/- 46.6 x 10(3)), respectively (P < 0.01). The IOD of P-gp and beta-catenin, which was 57.7 +/- 75.5 x 10(3)) and 32.0 +/- 47.0 x 10(3)) respectively, was significantly lower in ESCC than in normal mucosa, which was 114.8 +/- 106.6 x 10(3)) and 46.1 +/- 35.7 x 10(3)), respectively (P < 0.01). According to the following order, well differentiated moderately differentiated poorly differentiated, the IOD of MRP2 increased in ESCC (P < 0.01). The IOD of beta-catenin was higher in poorly differentiated ESCC than in well or moderately differentiated ESCC (P < 0.01). The IOD of Bcl-2 was lower in well differentiated ESCC than in poorly and moderately differentiated ESCC (P < 0.01). The IOD of beta-catenin and Bcl-2 was higher in the ESCC of specimens with infiltration depths that were in membrane mucosa than those in the muscular layer or serous coat (P < 0.01). The IOD of Bcl-2 was significantly higher in cases with lymph node metastasis than in cases without (P < 0.01). Positive correlations which were respectively between the expressions of P-gp and MRP2, the expressions of P-gp and Bcl-2 were found (r = 0.288 and r = 0.253, respectively, P < 0.01).

CONCLUSIONSMRP2, P-gp, and Bcl-2 may be taken as prognostic factors for MDR of ESCC. beta-catenin may play an important role in carcinogenesis and progression of ESCC.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Differentiation ; Esophageal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Multidrug Resistance-Associated Proteins ; metabolism ; Neoplasm Invasiveness ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Young Adult ; beta Catenin ; metabolism

Background and Objective:As chemotherapy is generally used in the clinical treatment of cancer,the problem of multidrug resistance(MDR)of tumors against the chemotherapeutic agents becomes more and more serious.It has been the major cause for the failure of the chemotherapy.We detected the expressions of β-catenin and tumor drug resistance related proteins,MRP2,P-gp,and Bcl-2,in esophageal squamous cell carcinoma (ESCC)to explore their function and correlation in the occurrence and development of MDR in ESCC.Methods:We used the tissue microarray technique,immunohistochemistry,and image analysis methods to detect the expressions of MRP2,P-gp,β-catenin,and Bcl-2 proteins and analyze their relationships with clinical data in a ESCC tissue microarray consisting of 582 specimens of ESCC,294 specimens of normal mucosa,92 specimens of basal cell hyperplasia,and 87 specimens of dysplasia adjacent to cancer tissue.Results:The integral optical density(IOD)of MRP2 and Bcl-2,which was 195.7±175.9(×10~3)and 90.5±112.5(×10~3),respectively,was significantly higher in ESCC than in normal mucosa.which was 1 04.8±86.1(×10~3)and 25.2±46.6(×10~3),respectively(P＜0.01).The IOD of P-gp and β-catenin,which was 57.7±75.5(×10~3)and 32.0±47.0(×10~3)respectively,was significantly lower in ESCC than in normal mucosa,which was 114.8±106.6(×10~3)and 46.1±35.7(×10~3),respectively(P＜0.01).According to the following order.well differentiated-moderately differentiated-poorly differentiated.the 1OD of M RP2 increased in ESCC(P＜0.01).The IOD of β-catenin was higher in poorly differentiated ESCC than in well or moderately differentiated ESCC(P＜0.01).The IOD of Bcl-2 was lower in well differentiated ESCC than in poorly and moderately differentiated ESCC(P＜0.01).The IOD of β-catenin and Bcl-2 was higher in the ESCC of specimens with infiltration depths that were in membrane mucosa than those in the muscular layer or serous coat(P＜0.01).The IOD of Bcl-2 was significantly higher in cases with Iymph node metastasis than in cases without (P＜0.01).Positive correlations which were respectively between the expressions of P-gp and MRP2,the expressions of P-gp and Bcl-2 were found(r=0.288 and r=0.253.respectively,P＜0.01).Conclusions:MRP2,P-gp,and Bcl-2 may be taken as prognostic factors for MDR of ESCC.β-catenin may play an important role in carcinogenesis and progression of ESCC.

This study was aimed to develop and establish an efficient method for high through-put automatically extracting genomic DNA from EDTA-anticoagulated whole blood samples, and to utilize this method in routine rSSO HLA genotyping by luminex flow array assay, the genomic DNA was extracted automatically from 400 microl blood samples by using TECAN DNA workstation and 96-well plate with 2 ml volume per well. The yield and purity of each DNA sample was tested by UV-spectrophotometer, the integrity of these DNA samples were run electrophoresis on the agarose gel. Each DNA sample was subjected to PCR amplification and hybridization using One lambda rSSO HLA-A, -B and -DRB1 commercial kit, the fluorescent intensity for positive bead and negative bead hybridized with HLA-A, -B and -DRB1 PCR products were calculated and analyzed. The results showed that the mean yield and purity (A260/A280) of genomic DNA extracted from 400 microl whole blood samples were 3.217+/-0.715 microg and 1.710+/-0.103 respectively. The molecular weight was more than 15 kb in size and the fluorescent intensity for positive bead hybridized with HLA-A, -B and -DRB1 PCR products of each sample was >600 RFU, however, the fluorescent intensity for negative bead for each sample was <50 RFU. It is concluded that the highly qualified genomic DNA can be extracted automatically from blood samples of marrow-donors by using TECAN DNA workstation, and the extracted DNA samples are suitable for high through-put HLA genotyping by luminex flow array assay and other downstream transplant immunological and molecular biological experiments.
Biological Specimen Banks ; Bone Marrow ; DNA ; isolation & purification ; DNA Primers ; Genotype ; HLA Antigens ; genetics ; High-Throughput Screening Assays ; methods ; Humans ; Living Donors ; Nucleic Acid Hybridization ; methods ; Oligonucleotide Array Sequence Analysis ; methods

A 49-year-old woman visited the clinic because of acute hepatitis and acute kidney injury with decreased urine output presenting microscopic hematuria and proteinuria. An abdominal computed tomography revealed a localized, hypoattenuated lesion in a hepatic lateral segment, and kidney biopsy showed oxalate crystal deposition with tubular necrosis. In addition, the patient's 24-hour urinary excretion of oxalate was increased. Her kidney and liver injury improved after sessions of hemodialysis, and urinary oxalate excretion was normalized. Major mutations in primary hyperoxaluria have not been proven. A full sequencing of target genes may be helpful to diagnose a rare form of primary hyperoxaluria.
Acute Kidney Injury ; Biopsy ; Female ; Hematuria ; Hepatitis ; Humans ; Hyperoxaluria* ; Hyperoxaluria, Primary ; Kidney* ; Liver* ; Middle Aged ; Necrosis ; Proteinuria ; Renal Dialysis

Objective To work out the suitable iodine content in iodized salt among general population in Enshi Autonomous prefecture,Hubei province by determination of the iodine content in salt.Methods The method of direct titration was used to determine the iodine content in salt samples collected from residents in natural villages sampled from four directions of east,west,south and north in each township which was sampled from five directions of east,west,south,north and center in each city(county) in Enshi Autonomous prefecture,and salt samples were collected in Hubei Salt Industry Group Co.,Limited.Enshi Branch in 2011.The method of three-days weighing was used to estimate the resident's daily per capita intake of iodized salt.The appropriate iodine content for general population in salt was worked out according to the iodine content in salt from households and enterprises in Enshi Autonomous prefecture,the amount of iodine loss in iodized salt,the amount of per capita daily intake of iodized salt and the national iodine nutrition monitoring results.Results The median of iodine content in salt from residents and the production enterprises in 2011 was 33.5 mg/kg and 34.7 mg/kg,respectively.The residents' per capita salt intake was 10.9 g,actual intake of iodine wss 335.0 μg/d.Iodine content in iodized salt was 20 mg/kg ±30％ for the general population,actual intake of iodine was 149.4-250.4 μg/d.Conclusions The residents iodine intake is higher in Enshi Autonomous prefecture.Considering the comprehensive factors,including food iodine,water iodine,and iodine cooking loss,that affect the intake of salt iodine,the appropriate iodine content in iodized salt is 20 mg/kg ± 30％ for the general population.

BACKGROUND: Many studies have showed that apoptosis of endothelial cells plays a curial role in the progress of sepsis. But the role of simvastatin in apoptosis of endothelial cells induced by sepsis is not clear. The present study aimed to investigate the role of simvastatin in apoptosis of endothelial cells induced by sepsis and its mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were randomly divided into three groups: control group, sepsis serum intervention group (sepsis group) and simvastatin+sepsis serum intervention group (simvastatin group). After 24-hour incubation with corresponding culture medium, the relative growth rate of HUVECS in different groups was detected by MTT assay; the apoptosis of HUVECs was detected by Hoechst33258 assay and flow cytometry; and the expression of the Bcl-2 and Bax genes of HUVECs was detected by PCR. RESULTS: Compared with the sepsis group, HUVECs in the simvastatin group had a higher relative growth rate. Apoptotic HUVECs decreased significantly in the simvastatin group in comparison with the sepsis group. Expression of the Bcl-2 gene in HUVECs decreased obviously, but the expression of the Bax gene increased obviously after 24-hour incubation with sepsis serum;however, the expression of the Bcl-2 and Bax genes was just the opposite in the simvastatin group. CONCLUSIONS: Our study suggests that simvastatin can inhibit apoptosis of endothelial cells induced by sepsis through upregulating the expression of Bcl-2 and downregulating Bax. It may be one of the mechanisms for simvastatin to treat sepsis.

Objective　To observe the curative effect and sid e effect of the gastrokinetic agent mosapride citrate by RCT. Methods　 42 cases of functional dyspepsia (FD) were divided into two groups rando mly, the group of mosapride(21 cases):orally administrated mosapride, 5mg, t.i.d for 4 weeks, and the control (21 cases):orally administrated domperidone, 5mg, t.i.d for 4 weeks. Symptoms and side effects were recorded before and at d 14, d 28 after administration of the medicines according to GCP and double blind pri ciple. Gastric empting test was also carried out in randomly selected patients. Results　Mosapride and domperidone were significantly effective on alleviating symptoms of the patient with FD. In mosapride treated group the half emptying time was shortened and the 120 min remain rate was reduced. No sid e effect was found. Conclusion　These results suggest that mosa pride 5 mg t.i.d. is effective and safe on alleviating symptoms of patients with FD and improving the ga stric empting time.

Objective To evaluate the effect of acupuncture(manual acupuncture,electroacu-puncture)on prevention and treatment of nausea and vomiting after general anesthesia.Methods By searching CNKI,VIP,China Biomedical Literature Database,Wanfang Medical,Embase,PubMed,Co-chrane Clinical Trial Registry,randomized controlled trial(RCT)on acupuncture combined with antiemetics for the prevention and treatment of nausea and vomiting after general anesthesia were performed.The retrieval period was from the establishment of the database to September 2022.RevMan 5.3 was used for statistical analysis.Results Fifteen studies involving 1 493 patients were included.There were 741 patients in the antiemetic group and 752 patients in the acupuncture combined antiemetic group.The incidence of postoperative nausea in the acupuncture combined with antiemetics group was significantly lower than that in the simple antiemetics group(OR=0.43,95％CI 0.34-0.54,P＜0.001).The incidence of postopera-tive vomiting in the acupuncture combined with antiemetics group was significantly lower than that in the simple antiemetics group(OR=0.55,95％CI 0.45-0.66,P＜0.001).Conclusion The incidence of postoperative nausea and vomiting was lower in patients treated with acupuncture(manual acupuncture,electroacupuncture)combined with antiemetics than with antiemetics alone.

Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.