Screening Quorum-sensing-associated sRNA and construction of its overexpression and deletion mutant in Pseudomonas aeruginosa / 医学研究生学报

ObjectiveQuorum-sensing (QS) and small regulatory RNA (sRNA) play key regulatory roles in many signaling cascades of Pseudomonas aeruginosa. To investigate whether sRNA is involved in P. aeruginosa QS system, screening QS system-related sRNA, and to construct sRNA overexpression and deletion strains of Pseudomonas aeruginosa for further study of sRNA function.MethodsSRNA associated with the QS system was screened by qPCR and RNA-sequencing (RNA-seq). The target gene were amplified by PCR and inserted into the overexpression vector pROp200 or the homologous recombination vector pGSM-MR, respectively. The connection reaction solution of pROp200-sRNA and pGSM-ΔsRNA was transformed into Escherichia coli DH5a and SM10lp, respectively. The recombinant vectors were identified by PCR. The pROp200-sRNA was transformed into PAO1 by heat shock method, and the pGSM-ΔsRNA was transferred from SM10lp to PAO1 by conjugation. SRNA overexpression and deletion strains were identified by PCR, DNA sequencing and qPCR, the determination of the growth curves and the pyocyanin levels of strains.ResultsFive QS -associated sRNA P26, P5316.1, P30, P34 and AmiL were successfully screened by RNA-seq and qPCR. PCR, DNA sequencing and qPCR showed that sRNA of AmiL, P30 and P34 overexpression and knockout were successful. Compared with wild-type strain, sRNA overexpression and knockout had no significant effect on bacterial growth curve. It were notably that overexpression of AmiL and P30 inhibited and increase the production of pyocyanin, respectively (P0.05).ConclusionThe sRNA overexpression and deletion strains have been successfully constructed and can be used to study the regulatory relationship between sRNA and QS systems, and to further functional study.

Signal molecule 3-oxo-C12-HSL of Pseudomonas aeruginosa promotes autophagy of mouse alveolar macrophages MH-S cells / 基础医学与临床

Objective To investigate the effect of 3-oxo-C12-HSL on autophagy in mouse alveolar macrophages MH-S cells. Methods MH-S cells were treated with culture supernatants of the mutant and wild type Pseudomonas aeruginosa(PA) strains of LasI gene(3-oxo-C12-HSL synthetic gene) and chemically synthesized 3-oxo-C12-HSL signaling molecules. GFP puncta was observed by laser confocal fluorescence microscopy and the ratio of LC3Ⅱ/LC3Ⅰ was detected by Western blot to detect the formation of autophagic.Autophagic flux was also detected by mo-nitoring the degradation of p62 and the change of chloroquine to LC3Ⅱ/LC3Ⅰratio. Results The supernatant of the culture medium of the wild type PA strain increased the GFP puncta of the MH-S cells(P<0.05) and the ra-tio of LC3Ⅱ/LC3Ⅰ(P<0.01),The mutant PA strain of LasI gene could not cause the above changes related to autophagy. The chemically synthesized 3-oxo-C12-HSL signal molecules could increase the number of autophagic bodies and the expression of LC3Ⅱ (P<0.01). Autophagic substrate p62 was degraded by 3-oxo-C12-HSL. Chloroquine, a lysosomal inhibitor, enhanced LC3Ⅱaccumulation caused by 3-oxo-C12-HSL (P<0.05,P<0.01).Conclusions 3-oxo-C12-HSL increases the level of autophagy in MH-S cells.

Efficacy of sublingual immunotherapy with dermatophagoides farinae drops in monosensitized and polysensitized patients with allergic rhinitis / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To compare the efficacy of sublingual immunotherapy (SLIT) with standardized Dermatophagoides farinae drops in monosensitized and polysensitized patients with allergic rhinitis.</p><p><b>METHODS</b>The efficacy of SLIT in 69 patients who were sensitized to house dust mites and treated with Dermatophagoides farinae drops for 1.5-2.0 year with complete clinical data were analyzed retrospectively. These patients had been divided into the monoallergen sensitized group and polyallergen sensitized group according to the results of skin prick tests. The total medication score (TMS) and the total nasal symptoms score (TNSS) were evaluated before and half an year, 1.0 year and 1.5-2.0 years after SLIT treatment.</p><p><b>RESULTS</b>After SLIT treatment for half an year, the TNSS in the monoallergen sensitized group (2.00 [1.00; 3.00]) was significantly lower than that in the polyallergen sensitized group (3.00 [2.00; 4.00], Z = -2.851, P < 0.05), this significant difference of TNSS between the two groups was also found after SLIT treatment for 1.0 year (0.00 [0.00; 1.00], 2.00 [0.00; 3.00], Z = -2.590, P < 0.05). Whereas, there was no significant difference between the two groups after 1.5-2.0 years treatment refer to the TNSS (0.00 [0.00; 1.00], 0.00 [0. 00; 2.00], Z = -1.461, P > 0.05). Half an year, 1.0 year and 1.5-2.0 years after SLIT treatment, the TMS in both groups reduced significantly, with no significant difference between two groups (Z value was - 0.777, -0.944, -0.907, all P > 0. 05).</p><p><b>CONCLUSIONS</b>SLIT with Dermatophagoides farinae drops is effective in monosensitized and polysensitized patients with allergic rhinitis. And equivalent efficacy could be achieved after 1.5-2.years.</p>

Human CD96 gene cloning, expression and identification / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct and express human CD96 gene outer membrane domain (hCD96om) in prokaryotic cells and prepare rabbit polyclonal antibody of hCD96om.</p><p><b>METHODS</b>hCD96om was amplified by RT-PCR from the peripheral blood of patients with acute myeloid leukemia and inserted into prokaryotic expression vector pET32a(+) to construct the recombinant plasmid pET32-CD96. The expression of hCD96om was induced by IPTG in BL21(DE3) cells, and the expression product was identified by Western blotting. The anti-hCD96 polyclonal antibody was prepared by immunization of rabbits with the fusion protein. The specificity of anti-hCD96 antibody was determined by Western blotting.</p><p><b>RESULTS</b>hCD96om protein was expressed in E.coli BL21(DE3) cells in the form of inclusion body, with a relative molecular mass around 37 kD. Western blotting showed a specific reaction of the prepared antiserum with the 70 kD protein extracted from human leukemia cell line HL-60 cells and with the 37 kD hCD96om fusion protein.</p><p><b>CONCLUSION</b>The CD96 gene of human has been successfully cloned and expressed in BL21(DE3) cells, and its rabbit polyclonal antibody has been obtained.</p>

Targeted blockage of RNA binding protein E2 by decoy RNA induces the granulocytic differentiation of K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To use a decoy RNA targeted blockage of the RNA binding protein E2 (hnRNP E2) resulting in the CCAAT/enhancer-binding protein alpha (C/EBP alpha) gene's abnormal translation and investigate its effect on the granulocytic differentiation of K562 cells and the probable molecular mechanism.</p><p><b>METHODS</b>The hnRNP E2 decoy RNA expression plasmid was constructed and transfected into K562 cells with cationic liposome, and stable expression cells were obtained by G418 selection. The changes of C/EBP alpha and granulocyte colony-stimulating factor receptor (G-CSFR) gene expression were detected by RT-PCR and Western blot. The morphologic changes were observed after Wright-Giemsa staining. The expression of granulocytic differentiation antigens CD13 and CD15 was studied by immunocytochemistry.</p><p><b>RESULTS</b>The stably expressed pG cells were obtained. Its C/EBP alpha mRNA level remained unchanged, while 42kD-C/EBP alpha protein expression was increased by (49.7 +/- 5.5)% (P < 0.05); and G-CSFR mRNA was increased by (42.1 +/- 3.6)% (P < .05), and its protein was increased by (37.4 +/- 6.2)% (P < 0.05) compared to that in the K562 control cells. The characteristics of polymorphonuclear neutrophils appeared in pG cells and CD13 and CD15 positive cell ratios were (18.7 +/- 2.5)% and (26.3 +/- 2.9)% respectively.</p><p><b>CONCLUSIONS</b>HnRNP E2 decoy RNA can induce granulocytic differentiation of K562 cells, and G-CSF promotes this effect. The mechanisms may be that decoy RNA specifically blocks hnRNP E2, hence regulates the translation of C/ EBP alpha mRNA, restores the expression of 42kD-C/EBP alpha, and then up-regulates the expression of G-CSFR gene.</p>

Targeted blockage of STAT5 by a decoy oligodeoxynucleotide inhibits the growth and proliferation of K562 cells / 中华血液学杂志

<p><b>OBJECTIVES</b>To investigate targeted blockage of BCR/ABL oncoprotein mediated cell transformation by STAT5 decoy oligodeoxynucleotide (ODN), its effect on the growth and proliferation inhibition of K562 cells and the related molecular mechanisms.</p><p><b>METHODS</b>STAT5 decoy ODN, designed and synthesized in vitro, was transfected into K562 cells by cationic lipid. The cell growth curve and colony formation assay were used to reflect the growth and proliferation capacity of K562 cells, RT-PCR to detect the expression of three genes downstream STAT5.</p><p><b>RESULTS</b>Confocal microscopy demonstrated that STAT5 decoy ODN was successfully transfected into K562 cells (95.2% positive cells). STAT5 decoy ODN inhibited the growth of K562 cells (inhibition rate 77.7%) and their colony formation capacity (Decoy ODN treated group 8.3% vs control group 35.7%, P < 0.05) after the treatment with STAT5 decoy ODN, the expressions of c-myc, bcl-X(L), cyclin D1 mRNA were down-regulated by 15.4%, 30.8%, 29.1%, respectively in the K562 cells.</p><p><b>CONCLUSIONS</b>STAT5 decoy ODN inhibits the growth and proliferation of K562 cells. The mechanisms may be that decoy ODN blocks the transcriptional activation potent of STAT5 and down-regulates the expression of these tumor related genes downstream STAT5.</p>

Quantification of bcr/abl mRNA in patients with chronic myeloid leukemia by using real-time quantitative fluorescence PCR with self-quenched primer / 中华检验医学杂志

Objective To establish a quantitative RT-PCR method with self-quenched fluorogenic probe for detection of bcr/abl mRNA in patients with chronic myeloid leukemia for providing a useful tool for diagnosis of CML,evaluation of therapeutic effect and monitoring of minimal residual disease(MRD). Methods bcr/abl gene from cultured K562 cells was amplified by conventional RT-PCR.The standard quantitative plasmid was constructed by A-T clone method.The self-quenched fluorogenic quantitative RT- PCR method(FQ-RT-PCR)for determination of bcr/abl mRNA was established successfully using the ABI PRISM 7000 PCR Detector.The linear range,sensitivity,stability,and repetitiveness of the method were determined.The marrow samples from 25 CML patients and 3 ALL patients were assessed.Results The sensitivity of the FQ-RT-PCR was 10 copies/?l recombined plasmid,and bcr/abl mRNA can be detected from 1 K562 cell in 10~5 normal cells.The linear range was 10~2-10~9 copies/?l recombined plasmid.The coefficient variation(CV)value was 2.1% in intra-assay and 6.1% in inter-assay.The median ber/abl mRNA expression level was 4.50?10~4 copies/?g RNA [(0.45-89.00)?10~4],5.45?10~4 copies/?g RNA [(2.95-19.30)?10~4 ],13.00?10~4 copies/?g RNA [(4.10-89.00)?10~4] and 2.35?10~4 copies/?g RNA [(0.45-5.12)?10~4] in 25 CML patients,11 patients in the incipient chronic phase,6 patients in blastic crisis,8 patients in chronic period after treatment,respectively.The bcr/abl mRNA level in blastic crisis was significantly higher than that in chronic phase(q= 3.41,P