Detection of IgG protein in human urine based on vertical flow paper microfluidic chip / 生物工程学报

The kidney is the body's most important organ and the protein components in urine could be detected for diagnosing certain diseases. The amount of IgG protein in urine could be used to determine the degree of kidney function damage. IgG protein in human urine was detected by vertical flow paper-based microfluidic chip, double-antibody sandwich immunoreaction, and cell phone image processing. The results showed that using an IgG antibody concentration of 500 μg/mL and a gold standard antibody concentration of 100 μg/mL, the image signal showed a good linear relationship in the range of IgG concentration of 0.2-3.2 μg/mL, with R2=0.973 3 achieved. A complete set of detection devices were designed and the detection method showed good non-specificity.

Establishment of a stable HEK293T cell line with c.392G>T (p.131G>V) mutation site knockout in gene using CRISPR/Cas9 technique / 南方医科大学学报

OBJECTIVE@#To establish a stable HEK293T cell line with c.392G>T (p.131G>V) mutation site knockout in gene using CRISPR/Cas9 technique.@*METHODS@#We designed 4 pairs of small guide RNA (sgRNA) for c.392G>T(p.131G>V) mutation site, and constructed exogenous PX458 plasmids expressing Cas9-sgRNA. The plasmids were transfected into HEK293T cells, and the cells expressing GFP fluorescent protein were separated by flow cytometry for further culture. After verification of the knockout efficiency using T7 endonuclease Ⅰ, the monoclonal cells were screened by limiting dilution and DNA sequencing to confirm the knockout. We detected the expressions of mRNA and protein and examined functional changes of the genetically modified cells.@*RESULTS@#We successfully constructed the Cas9-sgRNA exogenous PX458 plasmid based on the c.392G>T(p.131G>V) mutation site of gene. The editing efficiency of the 4 pairs of sgRNA, as detected by T7E1 enzyme digestion, was 6.74%, 12.36%, 12.54% and 2.94%. Sanger sequencing confirmed that the HEK293T cell line with stable knockout of c.392G>T(p.131G>V) was successfully constructed. The genetically modified cells expressed lower levels of mRNA and protein and showed reduced enzyme activity and proliferative capacity and increased apoptosis in response to vitamin K3 treatment.@*CONCLUSIONS@#We successfully constructed a stable HEK293T cell model with gene c.392G>T(p.131G>V) mutation site knockout to facilitate future study of gene repair.

Effect of infliximab combined with methylprednisolone on expressions of NF-κB, TRADD, and FADD in rat acute spinal cord injury.

STUDY DESIGN: The possibility to prevent acute spinal cord injury (ASCI) by selective infliximab combined with methylprednisolone (MP) was assessed in experimental ASCI. OBJECTIVE: To investigate the effects of infliximab, MP, and the combination of these 2 agents on expressions of NF-κB (nuclear factor Kappa B), TRADD (tumor necrosis factor receptor associated death domain), and FADD (fas associated death domain) in a rat model of acute spinal cord injury (ASCI), and to confirm the therapeutic efficacy and possible mechanism of infliximab combined with MP in the treatment of ASCI. SUMMARY OF BACKGROUND DATA: The theory that SCI can induce tumor necrosis factor-α expression at the injury site has been evaluated. However, there are few data to confirm the therapeutic efficacy of infliximab combinated with MP in the treatment of rat SCI METHODS: One hundred eighty adult male Sprague Dawley rats with 280 to 300 g body weight were allocated randomly and accordingly. We applied Basso, Beattie, Bresnahan locomotor rating scale to assess the hindlimb motor functional score (10 rats × 6 groups), the hematoxylin and eosin stain and immunohistochemistry stain (10 rats × 6 groups) to assay the morphological changes of spinal cord, the arrangement and expressions of NF-κB, TRADD and FADD, and the RT-PCR (10 rats × 6 groups) to evaluate the messenger RNA expressions of NF-κB, TRADD, and FADD. RESULTS: The results showed that both infliximab and MP could lower the expressions of NF-κB, TRADD, and FADD 24 hours after the ASCI, and increased Basso, Beattie, Bresnahan score on the 14th and the 21st days after ASCI, suggesting possible neuroprotective effectiveness on attenuating the severity of neurological deficits and improving the locomotor function in the rat ASCI model. Moreover, infliximab combined with MP exhibited the more powerful ability to this amelioration. CONCLUSION: Infliximab combined with methylprednisolone may be an effective treatment for the recovery of ASCI. Further study is needed to determine if this neuroprotective effect is seen for long-term outcomes especially in human ASCI.

Effects of quercetin on proliferation of lung cancer cell line A549 by down-regulating hTERT gene expression / 第三军医大学学报

Objective To investigate the effects of quercetin on the growth of lung cancer cell line A549 and the expression of hTERT gene. Methods The number of viable cells was ascertained by trypan blue dye exclusion test. Morphological changes of apoptotic cells were observed by electronic microscopy and DNA ladder assay. The telomerase activity was analyzed by PCR-TRAP assay and hTERT mRNA expression was detected by quantitative RT-PCR. Results Quercetin had a significant inhibition on the proliferation of A549 cells in a dose-dependent manner. The IC50 was 22.5 ?mol/L after exposure to quercetin for 48 h. The results from electron microscopy and DNA ladder showed that apoptosis occurred in the A549 cells of treatment groups. The results of quantitative RT-PCR and PCR-TRAP revealed that the expression of hTERT mRNA was significantly inhibited by quercetin and telomerase activity was decreased. Conclusion Quercetin inhibits the growth of lung cancer cell line A549 in a dose-dependent manner,and induce their apoptosis. The down-regulated expression of hTERT,suppression of telomerase activity and destruction of telomere stability may all contribute to the mechanism of apoptosis induction.

Experimental study on shRNA targeted hTERT gene to suppress bladder cancer cell growth / 中华泌尿外科杂志

Objective To explore the mechanism of siRNA targeted hTERT gene to inhibit bladder cancer T24 cell growth by decreasing c-myc and TGF-?1 expression. Methods shRNA-hTERT-pTZU6+1 vectors were constructed by RNAi-DNA vector technique, then the vectors were transfected into bladder cancer T24 cells,and the most effective vector and its optimal concentration were screened using RT-PCR to detect hTERT expression in T24 cells.The T24 cell growth, the alternative of cell phase,the expression of hTERT,c-myc and TGF-?1 were detected by flow cytometry,RT-PCR and immunohistochemistry. Results Three shRNA-hTERT-pTZU6+1 vectors were successfully constructed.The most effective vector was ph2-shRNA vector,and its optimal concentration was 1.0 ?g.This vector decreased the cell growth and the cell number of S phase from 65.2% to 38.6%,increased the cell number of G0/G1 phase from 32.0% to 57.9%,and attenuated both mRNA and protein expressions of hTERT,c-myc and TGF-?1 in T24 cells. Conclusions Targeted hTERT gene with siRNA may inhibit the cell proliferation of bladder cancer;down-regulating hTERT expression by attenuating the expression of c-myc and TGF-?1 is probably involved in the mechanism.

Regulation of hTERT promoter transcription activity by bHLH family genes / 中国病理生理杂志

AIM: To investigate the transcription regulation of the promoter of human telomerase reverse transcriptase (hTERT) by transcription factors c-myc and [STBX]mad1. METHODS: The various plasmids including wild type hTERT (Tw) or mutant type hTERT (Td) which both harboring luciferase gene, the expression plasmids of c-myc and [STBX]mad1, and their control vectors were constructed. The plasmids were co-trans fected into bladder cancer cell lines T24, EJ and control cells COS-7 or fibrocytes by DOTAP liposome in various combining manner, respectively. The reporter gene luciferase activities in various groups were measured 48 h after transfection. RESULTS: The luciferase activities in T24 and EJ cells treated with Tw were much higher than that in COS-7 and fibrocytes cells treated with Tw, as well as higher than that in T24 and EJ cells treated with Td, respectively. In bladder cancer T24 and EJ cells, transcription factor c-myc and [STBX]mad1 positively and negatively regulated Tw expression in a dose-dependent manner. However, the effects of c-myc and [STBX]mad1 on Td were completely opposite to Tw. Combined with c-myc and [STBX]mad1, down-regulation of Tw expression was observed. CONCLUSION: c-myc and [STBX]mad1 regulates the transcriptional activity of hTERT promoter in bladder cancer cells, and the effects might highly depend on the conservative E-box sequence CACGTG.