[Role of allograft inflammatory factor-1 in regulating the proliferation, migration and apoptosis of colorectal cancer cells].

OBJECTIVE: To investigate the role of allograft inflammatory factor-1 (AIF-1) in colorectal cancer (CRC) progression and explore the possible mechanism. METHODS: The expression levels of AIF-1 in 70 CRC tissues and paired adjacent tissues were detected using immunohistochemistry and Western blotting, and the correlation of AIF-1 expression with the clinicopathological features of the patients was analyzed. In the CRC cell line SW480, the functional role of AIF-1 in regulating tumor progression was investigated by transfecting the cells with an AIF-1-overexpressing plasmid (AIF-1) and a negative control plasmid (NC). EdU proliferation assay and flow cytometry were used to assess the cell proliferation and cell cycle changes; Transwell migration assay and Annexin V-APC/7-AAD apoptosis assay kit were used to analyze the cell migration and apoptosis. The changes in the biological behaviors of the cells were observed after application of SB203580 to block the p38 MAPK pathway. The expression levels of CDK4, cyclin D1, P21, P27, MMP2, MMP9, Bax, Bcl2, Bcl-xl, p38 and p-p38 were detected using Western blotting. RESULTS: AIF-1 was down-regulated in CRC tissues compared with the adjacent normal tissues, and its expression level was positively correlated with lymph node metastasis (P=0.008), TNM stage (P=0.003) and tumor size (P=0.023). Overexpression of AIF-1 in SW480 cells significantly reduced EdU-positive cells and caused obvious cell cycle arrest in G1 phase (P<0.05). AIF-1 overexpression resulted in significantly lowered protein expressions of CDK4 and cyclin D1, enhanced expressions of P21 and P27, attenuated cell migration ability (P<0.001), and decreased protein levels of MMP2 and MMP9. AIF-1 overexpression also induced obvious apoptosis of SW480 cells (P<0.01), significantly increased the protein levels of Bax and p-p38, and decreased the protein levels of Bcl-2 and Bcl-xl; SB203580 significantly attenuated the apoptosis-inducing effect of AIF-1 overexpression. CONCLUSION: AIF-1 plays the role of a tumor suppressor in CRC by inhibiting cell proliferation, suppressing cell migration and inducing cell apoptosis. AIF-1 overexpression promotes the apoptosis of CRC cells by activating the p38 MAPK pathway.

[Effect of quercetin on neural stem cell proliferation in the subventricular zone of rats after focal cerebral ischemia-reperfusion injury].

OBJECTIVE: To study the effect of quercetin on the proliferation of neural stem cells in the subventricular zone (SVZ) of rats after focal cerebral ischemia. METHODS: An adult rat model of middle cerebral artery occlusion (MCAO) model was established by placement of an intraluminal filament at the origin of the MCA. Quercetin was administered intraperitoneally in the rats at a dose of 50 mg/kg every 3 days starting at 6 h after MCAO, and BrdU (50 mg/kg daily) was also injected intraperitoneally starting at 4 h after MCAO. BrdU-positive cells in the SVZ were counted at 7, 14 and 21 days after MCAO. RESULTS: Compared with the sham-operated group, the rats in the ischemic model group showed significantly increased BrdU-positive cells in the ipsilateral SVZ 7 days after MCAO, reaching the peak level on day 14 and beginning to decrease on day 21 (P<0.05). The number of ipsilateral BrdU-positive cells in quercetin group was significantly greater than that in the model group on days 7, 14 and 21 (P<0.05), and maintained the high level on day 21. CONCLUSION: Quercetin can maintain a high level of neural stem cell proliferation in the SVZ after focal cerebral ischemia in adult rats.

[Protective effect of mild hypothermia on astrocytes with traumatic or ischemic injury].

OBJECTIVE: To investigate the protective effect of mild hypothermia on rat astrocytes with traumatic or ischemic injury. METHODS: Rat astrocytes in primary culture were subjected to scratching or hypoxic injury and exposed to normothermia (37 degrees celsius;) or hypothermia (34 or 32 degrees celsius;) for 24 h. The morphology of the astrocytes was evaluated by live/dead staining, and the cell injury was measured by lactate dehydrogenase (LDH) release assay. RESULTS: As the temperature reduced the LDH release rate from the cells in hypoxic group decreased significantly, to (11.48 - or + 1.53)% at 34 degrees celsius; and (3.79 - or + 0.45)% at 32 degrees celsius; as compared to that in normothermia [(33.02 - or + 3.58)%] in the absence of rat white blood cells (WBC) (P<0.001). LDH release rate of the hypoxic cells further decreased in the presence of rat WBC to (51.14 - or + 2.17 )% at 37 degrees celsius;, (19.53 - or + 4.37)% at 34 degrees celsius; and (16.68 - or + 1.47)% at 32 degrees celsius; (P<0.001). In the scratched cells, with or without WBC, LDH release rate showed no significant variation between the 3 temperatures (P>0.05). CONCLUSION: Mild hypothermia offers obvious protective effects on rat astrocytes against ischemic damage but not against mechanical injury.

[Construction of delta-pIRES2-EGFP plasmid and its expression in HEK293 cells].

OBJECTIVE: To construct the delta-pIRES2-EGFP plasmid and investigate its expression in HEK293 cells. METHODS: Full length cDNA of rat delta opioid receptor gene amplified from rat brain tissues using reverse transcription and nested PCR was cloned into pMD20 T vector. The delta cDNA was inserted into pIRES2-EGFP plasmid to construct the recombinant eukaryotic plasmid delta-pIRES2-EGFP, which was transfected into HEK293 cells via Lipofectamine2000. The expression of delta was examined under fluorescence microscope. RESULTS: The recombinant delta-pIRES2-EGFP plasmid was successfully constructed, and high expression of delta was detected in HEK293 cells transfected by the plasmid. CONCLUSION: delta-pIRES2-EGFP has been successfully cloned, which shows high expression of delta in HEK293 cells.

[Construction and identification of cDNA library of E.coli mRNA with poly(A) tracts].

OBJECTIVE: To construct and identify the cDNA library of E.coli mRNA with poly(A) tracts. METHODS: The cDNA library of E.coli was constructed by restriction display-PCR (RD-PCR) technique, followed by sequencing and bioinformatics analyses. RESULTS: cDNA library of E.coli mRNA with poly(A) tracts was successfully constructed, and 66 gene fragments were sequenced. CONCLUSION: The constructed cDNA library of E.coli mRNA with poly(A) tracts contains a low rate of repetition and is of high quality.

[Cloning E. coli mRNAs with poly (A) tails by restriction digest polymerase chain reaction].

OBJECTIVE: To investigate the polyadenylation at the 3' terminal of the mRNAs in E.coli. METHODS: mRNAs of E.coli was enriched from total RNA with oligo (dT)-cellulose, and reverse transcription was performed using oligo(dT)18 as primer prior to synthesis of double strands cDNA which was digested with Sau 3A I to produce multiple gene fragments that were then ligated with adapters. Restriction digest-polymerase chain reaction (RD-PCR) was employed to divide the fragments into 10 groups using 10 different combinations of the 4 primers, and the products were cloned into T-vectors. RESULTS: More than 100 gene fragments were cloned, 30 of which were sequenced. CONCLUSION: Polyadenylation of E.coli mRNA is not a biochemical curiosity, and very likely, it is a general attribute of the mRNAs of bacteria.

A method for evaluation of the quality of DNA microarray spots.

To establish a method to evaluate the quality of the printed microarray and DNA fragments' immobilization. The target gene fragments that were made with the restriction display PCR (RD-PCR) technique were printed on a superamine modified glass slide, then immobilized with UV cross-linking and heat. This chip was hybridized with universal primers that were labeled with cy3-dUTP, as well as cDNA that was labeled with cy3-dCTP, as the conventional protocol. Most of the target gene fragments on the chip showed positive signals, but the negative control showed no signal, and vice versa. We established a method that enables an effective evaluation of the quality of the microarrays.