Development of national standards for genomic titer determination of recombinant type 5 adeno-associated virus / 中国生物制品学杂志

@#ObjectiveTo develop a national standard for genomic titer determination of recombinant type 5 adeno-associated virus(rAAV5).MethodsThe rAAV5-GFP stock solution prepared by the three-plasmid system was identified and verified for the appearance,pH,sterility,genomic titer,purity and infection titer according to the relevant requirements of Chinese Pharmacopoeia(Volume Ⅲ,2020 edition),which was diluted and subpackaged to prepare candidate standards according to the results;The stability of candidate standards was investigated by thermal acceleration test;Three laboratories were organized to collaboratively calibrate the candidate standards using droplet digital PCR(ddPCR).ResultsAll the detection indexes of the candidate standard and the stock solution met the relevant requirements;The genomic titer showed no significant decrease at 25,4,-20,-40,-80 ℃ for 1,3,4,6 months;Through collaborative calibration by three laboratories,the candidate standard was assigned a value of 2. 56 × 10(12)copies/mL,and the 95% confidence interval was 2. 48 ×10(12)copies/mL,and the 95% confidence interval was 2. 48 ×10(12)copies/mL ～ 2. 64 × 10(12)copies/mL ～ 2. 64 × 10(12)copies/mL.ConclusionThe developed national standard for the determination of rAAV5 genomic titer had good stability and might be used for the quality evaluation of rAAV5 related products.

Application of Duplex Droplet Digital PCR Detection of miR-888 and miR-891a in Semen Identification / 法医学杂志

OBJECTIVES@#To establish a system for simultaneous detection of miR-888 and miR-891a by droplet digital PCR (ddPCR), and to evaluate its application value in semen identification.@*METHODS@#The hydrolysis probes with different fluorescence modified reporter groups were designed to realize the detection of miR-888 and miR-891a by duplex ddPCR. A total of 75 samples of 5 body fluids (including peripheral blood, menstrual blood, semen, saliva and vaginal secretion) were detected. The difference analysis was conducted by Mann-Whitney U test. The semen differentiation ability of miR-888 and miR-891a was evaluated by ROC curve analysis and the optimal cut-off value was obtained.@*RESULTS@#There was no significant difference between the dual-plex assay and the single assay in this system. The detection sensitivity was up to 0.1 ng total RNA, and the intra- and inter-batch coefficients of variation were less than 15%. The expression levels of miR-888 and miR-891a detected by duplex ddPCR in semen were both higher than those in other body fluids. ROC curve analysis showed that the AUC of miR-888 was 0.976, the optimal cut-off value was 2.250 copies/μL, and the discrimination accuracy was 97.33%; the AUC of miR-891a was 1.000, the optimal cut-off value was 1.100 copies/μL, and the discrimination accuracy was 100%.@*CONCLUSIONS@#In this study, a method for detection of miR-888 and miR-891a by duplex ddPCR was successfully established. The system has good stability and repeatability and can be used for semen identification. Both miR-888 and miR-891a have high ability to identify semen, and the discrimination accuracy of miR-891a is higher.

Quantitative detection of the KRAS gene mutation in circulating tumor DNA for patients with colorectal cancer / 基础医学与临床

Objective To establish a liquid biopsy technique of KRAS gene G12D mutation and to assess its diagnostic value. Methods KRAS G12D mutation was analyzed by ddPCR in plasma DNA from 52 colorectal cancer patients and compared that of to 80 healthy subjects. KRAS gene sequencing in cancerous tissue of colorectal cancer patient being set as a golden standard, we evaluated the accuracy of ddPCR and analyzed the correlation between G12D mutation rate, plasma concentration;and their clinical manifestations in CRC. Results ddPCR indicated that KRAS G12D mutation rate and concentration(26.92％, 81.5 copies/mL) in the plasma samples of colorectal cancer patients were significantly higher than that of healthy subjects (8.75％, 16 copies/mL). Colorectal cancer patients with highly differentiated adenocarcinoma showed a significantly higher number of mutant copies than medium and low differentiated adenocarcinoma(P＜0.05);M2 patients had a significantly higher number of mutant copies than N1 and NO patients (P＜0.05);The concordance rate of KRAS gene mutation between cancerous tissue and plasma ctDNA was 87.50％ in CRC.Conclusions ddPCR is a fast, noninvasive and accurate method for plasma testing of ctDNA, and the test results could be used to monitor the course of the disease and as clinical guidelines.

Effects of berberine on mRNA expression levels of PPARγ and adipocytokines in insulin-resistant adipocytes / 中国中药杂志

Adipocytokines are closely associated with insulin resistance (IR) in adipose tissues, and they are more and more seriously taken in the study of the development of diabetes. This experiment was mainly to study the effect of berberine on mRNA expression levels of PPARγ and adipocytokines in insulin resistant adipocytes, and investigate the molecular mechanism of berberine in enhancing insulin sensitization and application advantages of droplet digital PCR (ddPCR). ddPCR absolute quantification analysis was taken in this experiment to simply and intuitively determine the appropriate reference genes. ddPCR and quantitative Real-time PCR (qPCR) were used to compare the effect of different doses of berberine (10, 20, 50, 100 μmol•L⁻¹) on mRNA expression levels of PPARγ, adiponectin, resistin and leptin in IR 3T3-L1adipocytes. Antagonist GW9662 was added to study the inherent correlation between PPARγ and adiponectin mRNA expression levels. ddPCR results showed that the expression level of β-actin in adipocytes was stable, and suitable as reference gene for normalization of quantitative PCR data. Both of ddPCR and qPCR results showed that, as compared with IR models, the mRNA expression levels of adiponectin were decreased in the treatment with berberine (10, 20, 50, 100 μmol•L⁻¹) in a dose-dependent manner (P<0.01); the expression of PPARγ was decreased by 20, 50, 100 μmol•L⁻¹ berberine in a dose-dependent manner in qPCR assay (P<0.01) and decreased only by 50 and 100 μmol•L⁻¹ berberine in ddPCR assay (P<0.05). PPARγ specific antagonist GW9662 intervention experiment showed that adiponectin gene expression was directly relevant with PPARγ (P<0.05). ddPCR probe assay showed that various doses of berberine could significantly reduce mRNA expression levels of resistin and leptin (P<0.01) in a dose-dependent manner. In conclusion, berberine enhanced insulin sensitization effect not by up-regulating adiponect in expression of transcriptional level in PPARγ-dependent manner, but may by the elevated multimerization of adiponectin in the posttranslational regulation level. Berberine down-regulated the resistin and leptin expression levels, which could alleviate lipolysis and improve IR in adipocytes. ddPCR provided better sensitivity and linear range than qPCR, with obvious technical advantages for the detection of low abundance expression of target genes.

A Trial of Screening of Genes Involved in Odontoblasts Differentiation from Human Dental Pulp Stem Cells

This study investigated the genes involved in the differentiation of odontoblasts derived from human dental pulp stem cells (hDPSCs). hDPSCs isolated from human tooth pulp were validated by fluorescence activated cell sorting (FACS). After odontogenic induction, hDPSCs were analyzed investigated by Alizaline red-S staining, ALP assay, ALP staining and RT-PCR. Differential display-polymerase chain reaction (DD-PCR) was performed to screen differentially expressed genes involved in the differentiation of hDPSCs. By FACS analysis, the stem cell markers CD24 and CD44 were found to be highly expressed in hDPSCs. When hDPSCs were treated with agents such as beta-glycerophosphate (beta-GP) and ascorbic acid (AA), nodule formation was exhibited within six weeks. The ALP activity of hDPSCs was found to elevate over time, with a detectable up-regulation at 14 days after odontogenic induction. RT-PCR analysis revealed that dentin sialophosphoprotein (DSPP) and osteocalcin (OC) expression had increased in a time-dependent manner in the induction culture. Through the use of DD-PCR, several genes were differentially detected following the odontogenic induction. These results suggest that these genes may possibly be linked to a variety of cellular process during odontogenesis. Furthermore, the characterization of these regulated genes during odontogenic induction will likely provide valuable new insights into the functions of odontoblasts.

Cloning and Expression of Sodium-Bicarbonate Cotransporter Isoform in Rat / 대한해부학회지

The potassium depletion has remarkable and opposite effect on kidney and body growth and has affected the expression of the several ion transporters. Previously, Ahn et al. have reported that HK alpha 1 and 2 subunit gene were upregulated in the hypokalemic rat kidney. To clone the unreported genes expressed in potassium deficiency, differential display PCR-based cloning strategy was used in normal and potassium-depleted rat kidney and a novel gene was isolated. Sequence analysis with blast search program identified a cDNA clone encoding an isoform of kidney sodium bicarbonate cotransporter-1. The tissue and cellular expression pattern of this gene were investigated with Northern analyses and in situ hybridization histochemistry (ISH) in normal and hypokalemic rats. This novel transcript was highly expressed in kidney and brain and at lower levels in distal colon, urinary bladder, and heart but not in salivary gland, stomach, liver, and lung in normal rat. In potassium-depleted rat, this transcript was upregulated in kidney, brain, and distal colon. By ISH, cellular distribution of this gene was highly expressed in S3 segment of proximal tubule, distal convoluted tubule, and cortical collecting duct of kidney and lower third of intestinal glands of distal colon but at lower levels in cortical and medullary thick ascending limb and medullary collecting duct of kidney and middle third of intestinal glands of distal colon. From these results, this candidate gene may play an important role in HCO3-transport by these organs during potassium depletion.

Screening of Differentially Expressed Genes between PC12 Cells and A123.7 Cells

The cAMP-dependent protein kinase(PKA) is an intracellular enzyme with serine-threonine kinase activity that plays a key role in cell growth, differentiation, and apoptosis in eukaryotes. In order to understand the PKA signal transduction pathway regulating cell life cycle and identify its role, we focused on the characterization of up-/down-regulated genes by PKA using the differential display polymerase chain reaction. Seven differentially expressed sequence tags(DEST) have been obtained. Among these DESTs, 2DESTs were homologous to the sequence of genes from BLAST search result. KC1-5 DEST that was up-regulated in A123.7 cells was highly corresponded to mouse apoptosis-related gene(MA-3) or mouse mRNA for topoisomerase inhibitor suppressed(TIS). MA-3 was induced in various types of apoptosis, specially in NGF-deprived apoptotic PC12 cells, TIS was down-regulated in the RVC lymphoma cells incubated with topoisomerase inhibitor that induces DNA strand breakages. PG1-1DEST that was highly expressed in PC12 cells was corresponded to transposon Tn103'-end. Tnansposon Tn10 was up-regulated in differentiated myeloblastic ML-1 cells by 12-O-tetradecanoylphorbol-13-acetate. This study illuminates that MA-3/TIS was down-regulated by PKA activity, and transposon Tn10 was up-regulated by it.

Cloning of adriamycin-resistant related (arr) gene in an adriamycin-resistant L1210 variant

A partial fragment of novel sequence (arr, adriamycin-resistant related) was previously identified using the differential display (DD)-PCR technique with adriamycin-resistant L1210 variant (L1210AdR), which shows a typical multidrug resistant (MDR) phenotypes. The present research shows the isolation of full length arr cDNA sequence. To clone the full length cDNA of arr gene, DD-PCR fragments were subjected to 5'- and 3'-Rapid Amplification of cDNA End (RACE) method. The cloned arr cDNA consisted of 770 bases and contained an open reading frame of 153 bases, encoding a protein of 51 amino acid with the molecular mass of 4 kDa by in vitro translation reactions. Northern blot analysis showed that a 770 bases transcript arr gene was overexpressed in adriamycin-resistant L1210 variant, but not in the parent suggesting that the arr gene may be involved in the adriamycin-resistant phenotypes.