[Effect of Notch1 on extracellular matrix deposition in the renal tubulointerstitium of diabetes].

The aim of the present study was to observe the effects of Notch1 and autophagy on extracellular matrix deposition in renal tubulointerstitium of diabetes and to explore the mechanism. The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice). After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. Rat renal tubular epithelial cells NRK52E were cultured under normal glucose (NG) and high glucose (HG) respectively, and the expression of Notch1 and LC3 proteins were detected by Western blotting. Autophagosomes in NRK52E cells with overexpressed and knockdown Notch1 under NG and HG conditions were observed by confocal microscope, and the expression changes of Notch1, Collagen-I and III protein were detected by immunofluorescence. The results showed that the Notch1 and Collagen-III expressions were increased (P < 0.01) and the LC3 expression was decreased (P < 0.05) in db/db mice compared with db/m mice. In vitro, the Notch1 was increased (P < 0.01) and the LC3 expression was decreased significantly (P < 0.01) in NRK52E cells of HG group compared with NG group. There was no significant change of Notch1 and LC3 expression between the mannitol (MA) group and the NG group. Autophagy was decreased and extracellular matrix deposition was aggravated when Notch1 was overexpressed. In contrast, autophagy was increased and extracellular matrix deposition was relieved by knockdown of Notch1 under HG conditions. In conclusion, Notch1 protein expression was increased and autophagy was reduced in renal tissue of diabetes and renal tubular epithelial cells under HG. The extracellular matrix deposition in the renal tubulointerstitium was relieved by regulating autophagy after the knockdown of Notch1.

[Effect of Blocking PYR Complex Binding to DNA Site by Peptide Nucleic Acid on γ-Globin Gene Expression].

OBJECTIVE: To investigate the effect of blocking polypyrimidine complex binding to DNA site by using peptide nucleic acid (PNA) on γ-globin gene expression. METHODS: PYR-PNA, ß-PNA and RS-PNA (random sequence-PNA) were designed and synthesized, then were transfected into K562 cells with the cationic liposome lipofectamine 2000 used as vector. The expression of γ-globin gene at both the transcriptional and translational level was detected by RT-PCR and the Western blot respectively at 24 h, 48 h and 72 h after transfection with PNAs. RESULTS: Compared with RS-PNA and control groups, the expression of γ-globin gene at mRNA and protein levels in PYR-PNA group was significantly up-regulated(P<0.05), especially at 48 h after tranfection, the levels of mRNA and protein in PYR-PNA group were increased by 2.0 and 2.5 times than those in control group, respectively. CONCLUSION: PYR-PNA can significantly up-regulate the expression of γ-globin gene in K562 cells, this study may provide a new research idea for gene therapy of ß-thalassemia.

The Prevalence and Spectrum of α-Thalassemia in Guizhou Province of South China.

α-Thalassemia (α-thal) is one of the most prevalent genetic diseases in the world and is especially frequent in tropical and subtropical regions, including South China. The aim of this study was to investigate the prevalence and spectrum of α-thal in Guizhou Province as this information was unknown. A total of 40 α-thal carriers were determined in 1219 newborn umbilical cord blood samples by hemoglobin (Hb) electrophoresis combined with DNA analysis, which revealed that the carrier rate of α-thal in Guizhou Province was 3.28%. One thousand and forty-five individuals referred to our hospital were tested for α-thal mutations. Two hundred and twenty-four cases were determined as α-thal carriers or patients. A total of 11 genotypes and five different α-thal mutations were identified in these 224 cases. Of these mutations, more than 96.0% were deletions, including - -(SEA) (65.89%), -α(3.7) (rightward) (22.87%) and -α(4.2) (leftward) (7.74%). The other two nondeletional mutations, Hb Constant Spring (Hb CS, α(CS)α, HBA2: c.427T > C) and Hb Quong Sze [Hb QS, α(QS)α, HBA2: c.377T > C (or HBA1)] account for 2.71% and 0.78%, respectively. The results of this study will be useful in genetic counseling and prenatal diagnosis (PND) of α-thal in Guizhou Province.

Spectrum of ß-thalassemia mutations in Guizhou Province, PR China, including first observation of codon 121 (GAA>TAA) in Chinese population.

OBJECTIVES: Although the population prevalence and mutation spectrum of ß-thalassemia in most areas of south China have been characterized, the mutations have not been elucidated in Guizhou Province. The aim of this study was to investigate the spectrum of ß-thalassemia in this province. DESIGN AND METHODS: We detected and analyzed ß-globin gene mutations in 407 ß-thalassemia patients and carriers by PCR-based reverse dot blot (RBD) and direct sequencing methods. RESULTS: Twelve types of ß-globin gene mutations were detected. Among the 12 different mutations, six mutations are common, accounting for 97% of mutated alleles. The most prevalent mutation is codon 17 (A→T) with an allele frequency of 40.7%. In addition, codon 121 (GAA>TAA), a rare dominant mutation, was detected in a patient with ß-thalassemia intermedia for the first time in China. CONCLUSIONS: The results of this study will be useful in genetic counseling and prenatal diagnostic service of ß-thalassemia in Guizhou Province.

Upregulated functional expression of Toll like receptor 4 in mesenchymal stem cells induced by lipopolysaccharide.

BACKGROUND: The coordinated change of haematopoietic supporting microenvironment in bone marrow (BM) is crucial for innate immunity and inflammation. As the precursors of marrow stroma, BM derived mesenchymal stem cells (MSCs) promote haematopoietic function, but their roles in innate immunity or inflammation have not been investigated. Here we investigated the expression of Toll like receptor 4 (TLR-4) and the effect of lipopolysaccharide (LPS) on its expression in BM MSCs in vitro. METHODS: MSCs were harvested from adult rat's BM cells by density gradient centrifugation and adhesive culture. The purity of MSCs were identified with the cell morphological feature and osteogenic capacity, the phenotypes were tested by flow cytometry. Cultured MSCs were treated by LPS (1 microg/ml, 10 microg/ml or 100 microg/ml) for 24 hours. The relative expression levels of TLR-4 mRNA were detected by semiquantitative reverse transcription polymerase chain reaction and costimulatory molecules (CD80, CD86 and MHC-II) expressed on MSCs were analyzed by flow cytometry. The levels of tumor necrosis factor-alpha (TNF-alpha) in supernatants were determined by enzyme linked immunosorbent assay. RESULTS: After incubation with LPS, MSCs expressed the higher levels of TLR-4 mRNA, costimulatory molecules and TNF-alpha than the untreated group: LPS 10 microg/ml was the most effective (P < 0.01); the levels of TLR-4 mRNA, costimulatory molecules and TNF-alpha decreased when MSCs were exposed to 100 microg/ml LPS. Except for MHC-II and TNF-alpha (P > 0.05), the levels of CD80, CD86 and TLR-4 mRNA were significantly lower than that in the treated group of 10 microg/ml (P < 0.01). CONCLUSION: MSCs expressed TLR-4 mRNA. LPS activated the functional expression levels of TLR-4 in MSCs although the activity may depend on the concentration of LPS.

[The effect of lipopolysaccharide on the expression and activity of Toll-like receptor 4 in mesenchymal stem cells].

OBJECTIVE: To investigate the effect of lipopolysaccharide(LPS) on the expression and activity of Toll-like receptor 4(TLR-4) in mesenchymal stem cells (MSC). METHODS: MSCs were harvested from adult rats bone marrow cells by density gradient centrifugation and adhesive culture. The purity of MSC were identified with the cell morphology and osteogenic capacity. The phenotypes were assayed by flow cytometry. Cultured MSCs were treated by LPS with various concentration (1 microg/ml, 10 microg/ml or 100 microg/ml) for 24 hours. The relative expression levels of TLR-4 mRNA were detected by semiquantitative RT-PCR, and costimulatory molecules (CD80, CD86 and MHC-II) expressed on MSC were analyzed by flow cytometry. The levels of TNF-alpha in supernatants were determined by double-antibody sandwich ELISA. RESULTS: The expression levels of CD80, CD86, MHC-II, TLR-4 mRNA and TNF-alpha in MSC were (9.56 +/- 0.69)%, (22.03 +/- 2.03)%, (2.51 +/- 0.97)%, relative magnitude (0.61 +/- 0.10), (4.97 +/- 2.98) pg/ml, respectively. After incubation with LPS, MSC expressed higher levels of TLR-4 mRNA and costimulatory molecules, and the levels of TNF-alpha were higher than that in untreated group. Among the various concentration of LPS, 10 microg/ml emerged as the most effective in increasing the levels of TLR-4 mRNA (relative magnitude 1.55 +/- 0.02), costimulatory molecules [CD80 (41.70 +/- 2.92)%, CD86 (59.72 +/- 2.00)%, MHC-II (24.56 +/- 2.19)%] and TNF-alpha [(213.12 +/- 69.08) pg/ml] (P < 0.01). The levels of TLR-4 mRNA, costimulatory molecules and TNF-alpha began to decrease when MSC exposed to 100 microg/ml LPS (P < 0.05). Except for the levels of TNF-alpha [(158.05 +/- 28. 05) pg/ml] and MHC-II [(5.62 +/- 2.31)%] (P > 0.05), the levels of CD80, CD86, MHC-II and TLR-4 mRNA were significantly lower than the 10 microg/ml treatment group (P < 0.01). CONCLUSION: MSCs are able to express TLR-4 mRNA. LPS could activate the expression of TLR-4 in MSC obviously, but the activity is dependent on the specific concentration.