Isolation and culture of mouse adipose-derived stem cells and their homing to the intestinal tract / 中国组织工程研究

BACKGROUND:Adipose-derived mesenchymal stem cels have gained more and more attention due to their high safety, less invasiveness, easy purification and rapid proliferation. OBJECTIVE:To isolate and culture adipose-derived mesenchymal stem cels efficiently and rapidly with high purity and then to explore the cel homing to the intestinal tract after fluorescence labeling. METHODS: Mouse adipose tissue obtained from the groin and epididymis was asepticaly isolated and digested with 0.1% colagenase I. Then the digested cels and undigested adipose tissue were cultured together in the dish to harvest adipose-derived mesenchymal stem cels. The cels were identified by morphology, surface markers, growth kinetics, osteogenic and adipogenic differentiation potential, and then labeled by PKH67 before injected into ulcerative colitis mouse models through the tail vein to observe their homing to the intestinal tract. RESULTS AND CONCLUSION:In vitro, adipose-derived mesenchymal stem cels isolated by this method exhibited spindle-like appearance, grew intensively and arranged in a swirling shape. Adipose-derived mesenchymal stem cels expressed CD29, CD44 and CD90, but not expressed CD45. After osteogenic induction, alkaline phosphatase staining showed black particles and alizarin red S staining showed red mineralized nodules. After adipogenic induction, oil red O staining showed many lipid droplets were dyed red. Cel growth curve showed cels at 3-5 days were in logarithmic growth phase and they were active. Under fluorescence microscopy, frozen sections of the colon were found green fluorescence points that were increased with time. Results suggest that adipose-derived mesenchymal stem cels isolated and culturedin vitro can proliferate rapidly, purify easily and can be induced to osteoblasts and adipocytes; in vivo, PKH67-labeled cels can home to and proliferate in the colon.

Shortening of the 3' untranslated region: an important mechanism leading to overexpression of HMGA2 in serous ovarian cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Oncofetal protein high-mobility-group AT-hook protein 2 (HMGA2) is reactivated in serous ovarian cancer (SOC) and its overexpression correlates with poor prognosis. To explore the mechanism, we investigated whether HMGA2 could avoid microRNA regulation due to gene truncation or 3' UTR shortening by alternative polyadenylation.</p><p><b>METHODS</b>Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate the abundance of different regions of HMGA2 mRNA in 46 SOC samples. Rapid amplification of cDNA 3' ends (3' RACE) and Southern blotting were used to confirm the shortening of 3' untranslated region (UTR). 5' RACE and Southern blotting were used to prove the mRNA decay.</p><p><b>RESULTS</b>No significant difference in the ratio of the stable coding region to the fragile region was observed between SOC and control normal fallopian tubes, indicating that the HMGA2 gene is not truncated in SOC. Varying degrees of 3' UTR shortening in SOC samples were observed by comparing the abundance of the proximal region and distal region of the HMGA2 3' UTR. The ratio of the proximal to the distal region of the 3' UTR correlated significantly with expression of the HMGA2 coding region in SOC (r = 0.579, P < 0.01). Moreover, although the abundance of the HMGA2 coding region varied, all samples, including the very low expressed samples, exhibit relatively high levels of the proximal 3' UTR region, suggesting a dynamic decay of HMGA2 mRNA from the 5' end. The shortening of 3' UTR and the decay from the 5' end were confirmed by 3' RACE, 5' RACE and subsequent Southern blotting.</p><p><b>CONCLUSION</b>Heterogeneous 3' UTR lengths render HMGA2 susceptible to different levels of negative regulation by microRNAs, which represents an important mechanism of HMGA2 reactivation in SOC.</p>

Classified management of research reagents and consumables in hospitals / 中华医学科研管理杂志

To assist and improve the management level ofresearch reagents and consumables in hospitals.Research reagents and consumables can be classified into two groups the general and the special.Based on this classification and supported with informatization system,we can better serve researchers and improve the efficiency of research funds.Besides,the hospital can better control the consumption of research reagents and consumables,and supervise the use of research funds.

Increasing specificity of real time PCR to detect microRNA through primer design and annealing temperature increase / 北京大学学报(医学版)

Objective: To investigate the non-specific and inaccurate amplification in cases of highly similar sequences among family members and the length heterogeneity of mature microRNA ( miRNA) ,and find a condition that discriminates maximally among similar miRNA family members and detects the accurate expression level of miRNAs. Methods: Primers with their mismatches and/or 3' end at different positions were designed. Amplification efficiencies were compared using matched and various mismatched primers by RNA-tailing and primer-extension RT-PCR at different annealing temperatures. Expression levels of several miRNAs in mouse brain were compared using miRNA specific primers with different termini. Results: Raising annealing temperatures 12℃-14℃above the T_m of the primers maximally increased amplification specificity without sacrificing sensitivity. Primers designed with their termini on or near variant positions could efficiently discriminate between miRNA isoforms. Using primers that terminated before the end of the mature miRNA did not miss the detection of shorter mature miRNA and provided accurate expression levels. Conclusion: Careful primer design and higher annealing temperature can increase specificity and accuracy of real time PCR miRNA detection.

The effect of Heat shock protein 70 antiseuse oligonucleotides to the bladder cancer in mouse loaded with tumor / 肿瘤研究与临床

Objective To investigate The effect of Heat shock protein 70(HSP70) antisense oligonucleotides (ASO)to bladder carcinoma in mouse loaded with tumor.Methods The 40 mice loaded with tumor subcutaneously were established by cultured BIU-87 cells,and divided into 4 groups randomly when the subcutaneous neoplasms grew to about 100 mm3,namely,HSP70 mRNA ASO plus mitomycin C(MMC)group;HSP70 mRNA ASO group;MMC and blank control.HSP70 mRNA ASO were injected into neoplasms,10mmg/kg weight,twice every week,and MMC 0.1mg/kg weight,twice every week,and the above schemes were replaced with normal saline to blank.The neoplasms were peeled off,photograghed and weighed in 30 days.HSP70 expressions were examined with reverse transcription polymerase chain reaction (RT-PCR),mierovaseular density(MVD)was evaluated by immunohis to chemical staining and the tumor cells apoptosis was detected by terrainal deoxynucleotidyl transferase(TdT)-mediated dUTP-biotin nick end labeling technique (TUNEL).Results The tumor inhibition rate in ASO+MMC surpassed 50%.more than ASO or MMC respectively,and the differences were significantly(P＜0.05).The ASO and MMC exceeded blank group respectively(P＜0.05).The ASO was the same as the MMC(P＞0.05).The apoptotic index(AI)in ASO+MMC surpassed the other three groups (P＜0.05).The difference between ASO and MMC was not significant (P＞0.05),while the A1 of ASO or MMC was more than blank respectively(P＜0.05).The results of MVD were in accordance with the above results.Conclusion The injection of HSP70 mRNA ASO in tumor locally can inhibit neoplasm growth,and this effect might correlate with the inhibition of apoptosis and microvascular forming resulting from the ASO.

Profiling of microRNAs in mouse brain with real-time PCR array / 北京大学学报(医学版)

Objective:To examine global expression levels of microRNAs(miRNAs) in mouse cerebrum and to provide an important basis for detailed studies of individual miRNAs,their target genes,the miRNA-related regulatory networks in the mammalian central nervous system,and their implications in diseases.Methods:Low molecular weight RNA from cerebrum of five C57BL/6J mice were tailed and reverse transcribed by extended RT-primer.miRNA primers were carefully designed and arrayed on plates according to the Tm of each primer.PCR was carried out at different annealing temperatures using a gradient real-time PCR instrument.The relative expression level of each miRNA was calculated using 5sRNA for normalization.Results:Among the 285 miRNAs detected,260 were positive with varying abundance.Their frequency distribution was approximately a normal distribution.The expression levels of most miRNAs were in accordance with previously published results by microarray.However,the positive rate was higher than that detected by microarray.miRNAs originating from the same hairpin precursors expressed at similar or significantly different levels.Clusters of proximal miRNAs were similar or quite different in abundance.It is suggested that the fate of miRNA after transcription determined their abundance.Conclusion:Using the RNA-tailing and primer-extension PCR array method,we obtained expression profile of miRNA in mouse cerebrum,especially the relative expression data of many low abundant miRNA in mouse cerebrum,which will be of special help for studying the fine-tuning function of low-level miRNAs.

Real-time quantification of microRNAs by RNA-tailing and primer-extension RT-PCR / 北京大学学报(医学版)

Objective: To optimize and evaluate the modified RNA-tailing and primer-extension RT-PCR method in relative quantification of microRNAs (miRNAs) in several kinds of tissues. Methods: Small-sized RNAs (

Apoptin induces G_2-M arrest in cancer cells / 北京大学学报(医学版)

Objective: To further study the effect of apoptin in inducing cancer cell specific apoptosis and the possible applications in cancer therapy. Methods: Apoptin gene was amplified by PCR and inserted into pcDNA3.1(+) with a FLAG tag in front of the multi-cloning-site. Apoptin gene with the FLAG tag was sub-cloned into an adenovirus vector. Several cancer cell lines were transfected with pcDNA3. 1/FLAG/apoptin or infected with apoptin containing recombinant adenoviruses to study the morphologic changes. Ad/apoptin infected cells were also analyzed by flowcytometry after staining with PI. Results: Expressed apoptin was localized in the nucleus of cancer cells. Chromatin condensation occurred 2 or 3 days after Ad/apoptin(+) infection. Cell number in G 2-M phase increased dramatically after Ad/apoptin(+) infection. Conclusion: Apoptin can induce cell cycle G 2-M arrest and chromatin condensation in cancer cells.

Cell-cycle negative regulatory gene ANA is over-expressed in the brain tissues of patients with Down syndrome / 北京大学学报(医学版)

Objective: To investigate the expression level of genes located in chromosome 21 in the brain tissues of Down syndrome(DS). Methods: An optimized semi-quantitative RT-PCR method was used to evaluate the expression levels of seven genes encoded in chromosome 21 in fetal cortex brain and cerebellum of DS and the control at the end of 20 weeks of gestation. B2M was used as internal reference to normalize cell loss. Results: The expression levels of 6 genes in cortex and cerebellum, including DYRK1A, SYNJ1, PCP4, C21orf5, C21orf2 and C21orf106, were comparable between DS and the control. ANA, a cell-cycle negative regulatory gene, was over-expressed dramatically in the cortex but not in the cerebellum of DS. Conclusion: Over-expression of ANA may contribute to the reduction of neuronal density in DS brain.