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OBJECTIVE To establish the HPLC fingerprint of Xintongshu spray， determine the contents of identified components， and investigate the transferring patterns of the index components of decoction pieces， intermediates and spray， so as to provide scientific reference for technology management and quality control of Xintongshu spray. METHODS HPLC fingerprints of 13 batches of Xintongshu spray were established by the Similarity Evaluation System for Chromatographic Fingerprints of TCM （2012 edition）， and common peaks were identified； the contents of identified components were determined by HPLC. The paeonol in Moutan Cortex and ferulic acid in Chuanxiong Rhizoma were used as index components to investigate the transferring patterns of them in decoction pieces， intermediates and spray. RESULTS There were a total of 33 common peaks in the fingerprints of 13 batches of Xintongshu spray， and the similarities were more than 0.994. Eight components were identified， i.e. gallic acid （peak 5）， oxypaeoniflorin （peak 9）， chlorogenic acid（peak 10）， caffeic acid （peak 14）， paeoniflorin （peak 17）， ferulic acid （peak 21）， senkyunolide Ⅰ （peak 27） and paeonol （peak 31）. The contents of 8 components ranged from 0.590 3- 0.719 7， 0.565 7-0.851 3， 0.279 4-0.368 1， 0.080 6-0.106 1， 1.922 5-3.033 5， 0.151 3-0.191 6， 0.250 6-0.336 0， 3.056 7-4.161 0 mg/mL， respectively. The average transfer rates of paeonol and ferulic acid from decoction pieces to sprays were 63.76% and 38.06%， respectively. It was also found that the process in which the loss of paeonol was more than 30% was the extraction by percolation and negative pressure concentration of Moutan Cortex. The process in which the loss of ferulic acid was more than 50% was the steam distillation extraction process of Chuanxiong Rhizoma. CONCLUSIONS The established HPLC fingerprint and content determination method of Xintongshu spray are reproducible and specific. The key processes that cause a decrease in the average transfer rates of the index components are the extraction by percolation and negative pressure concentration of Moutan Cortex and steam distillation extraction of Chuanxiong Rhizoma.
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Objective:To construct a quantitatively diagnostic nomogram model by analyzing the clinical information of patients and the features of multi-modality ultrasound images of thyroid lesions, so as to preoperatively predict the malignant probability of suspicious thyroid nodules and provide effective references for clinical decision-making.Methods:A total of 933 patients, 1 121 thyroid nodules of C-TIRADS 3-5 categories, who underwent surgery in the Second Affiliated Hospital of Harbin Medical University from September 1, 2020 to June 10, 2021 were collected. The nodules were randomly divided into training ( n=897) and test groups ( n=224) in 8∶2 ratio. Finally, the diagnostic performance was evaluated by area under the curve (AUC). Results:①After preliminary screening by univariate analysis, multivariate analysis showed that age, echogenicity, orientation, echogenic foci, margin, posterior features, and elastic score were significantly correlated with benign and malignant nodules (all P<0.001), and the difference of halo between benign and malignant nodules was also statistically significant ( P=0.012). ②The AUC of nomogram was up to 0.903(95% CI=0.862-0.944) in the test set, and 0.889(95% CI=0.832-0.946) and 0.960(95% CI=0.925-0.994) in nodules with maximum diameter of ≤10 mm and of >10 mm respectively, which showed high diagnostic performance. Conclusions:The nomogram model could accurately differentiate malignant from benign thyroid nodules preoperatively, with the highest diagnostic performance for the nodules with maximum diameter of >10 mm, and effectively avoid the unnecessary fine-needle biopsy and surgical operation.
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Objective:To identify Down syndrome (DS) fetal encephalopathy related genes and signaling pathways via bioinformatics analysis, and to explore their potential mechanisms underlying the occurrence and development of DS neuropathology.Methods:Retrospective study.In December 2021, dataset GSE59630 was downloaded from the gene expression omnibus (GEO), and differentially expressed genes (DEGs) between DS and normal fetal brain tissue were identified by R software.Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and gene set enrichment analysis (GSEA) were performed on the genes identified.The protein-protein interaction (PPI) network was constructed based on search tool for the retrieval of interacting genes online database and Cytoscape software, and key modules and hub DEGs were identified.Real-time quantitative polymerase chain reaction technique was used to verify the expression of hub genes related to neurodegeneration in brain tissue of 3 pairs of DS and normal fetuses at the gestational age of 22-33 weeks.Results:A total of 225 DEGs were screened out from DS and normal fetal brain tissue, including 18 up-regulated genes and 207 down-regulated genes.GO functional enrichment analysis showed that DEGs were mainly enriched in neurogenesis, neuronal apoptosis, transcriptional regulation, mitochondrial energy metabolism, etc.KEGG pathway enrichment analysis revealed that DEGs were associated with a variety of neurodegenerative diseases.GSEA suggested that apoptosis and inflammatory responses play a vital part in the occurrence of DS neuropathology.Ten hub genes were identified by the PPI network established, and they were mainly related to histone acetylation and transcriptional regulation.According to the tissue verification result, the variations of RAB8A, TBP and TAF6 expression conformed to the microarray data. Conclusions:The key genes and signaling pathways identified by transcriptome analysis of fetal brain tissue facilitate the comprehensive understanding of the molecular mechanism of DS neuropathology.This study provides a novel insight into the clinical diagnosis and treatment of neurodevelopmental abnormalities and mental retardation in DS.
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Objective To discuss the application value of shear wave elastography (SWE) and superb microvascular imaging (SMI) in pathological grading of breast invasive ductal carcinoma (IDC).MethodsSixty patients with breast tumor were selected,which were totally 64 lesions.Before operation,SWE and SMI pattern were started to detect the lesions.And images in SWE and SMI pattern were saved.All lesions were proved to be IDC and pathological gradings were got according to the pathological result.The mean of Young′s modulus(AveT1) and maximum of Young′s modulus(AveT2) were recorded in SWE pattern,and degree of blood was recorded in SMI pattern followed Adler′s grading standard.Then differences of AveT1,AveT2 and degree of blood in different pathological grading of invasive ductal carcinoma were evaluated.Results ①In pathological grading 1 group,the average of AveT1 was (62.1±10.4)kPa,and the average of AveT2 was (93.0±20.1)kPa.In pathological grading 2 group,the average of AveT1 was (79.8±8.6)kPa,and the average of AveT2 was (120.6±18.7)kPa.In pathological grading 3 group,the average of AveT1 was (92.6±12.1)kPa,and the average of AveT2 was (137.3±21.5)kPa.The AveT1 and AveT2 were gradually growing with higher pathological grading of breast invasive ductal carcinoma,which were statistically significant (P<0.05).②In pathological grading 1 group,there were 7 cases in the degree of blood 0-Ⅰ and 14 in the degree of blood Ⅱ-Ⅲ.In pathological grading 2 group,there were 3 cases in the degree of blood 0-Ⅰ and 18 cases in the degree of blood Ⅱ-Ⅲ.In pathological grading 3 group,there were only 1 case in the degree of blood 0-Ⅰ and 21 cases in the degree of blood Ⅱ-Ⅲ.There were more lesions of rich blood with higher pathological grading of breast invasive ductal carcinoma,which was statistically significant (P<0.05).Conclusions There are differences of elastography and degree of blood in different pathological grading of breast invasive ductal carcinoma,SWE and SMI can prompt pathological grading and provide important clinical reference value.
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Objective:To determine the expression profile and potential roles of CD24 in oral squamous cell carcinoma and explore the values of CD24 function as a potential target of clinical therapy.Me-thods:Semi-quantitative immunohistochemistry was used to construct the expression profile of CD24 in 78 human oral tissues and 59 Hamster buccal pouch tissues.Real-time RT-PCR and Western blot were used to analyze the CD24 expression levels in oral DOK4 cells,oral cancer CAL-27 and WSU-HN6 cells. Then these two cancer cell lines were selected to evaluate the effect of all-trans retinoic acid (ATRA)and CD24 antibody on CD24 expression,and the proliferation and tumorsphere formation capacity of these two cell lines.Results:CD24 expression was found significantly elevated in both human and animal tissues compared with normal and benign tissues (P<0.05),as well as in oral cancer CAL-27 and WSU-HN6 cells compared with DOK cells (P<0.05).CAL-27 and WSU-HN6 cells possess increased proliferative and specific tumorsphere formation capability compared with DOK cells (P<0.05 ).Both ATRA and CD24 antibody were able to effectively inhibit the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells (P<0.05).Among them ATRA at least involved partially in the proliferation by down-regulating the CD24 expression (P<0.05 ),while CD24 antibody blocking had no effect on the CD24 expression.Conclusion:CD24 was upregulated in oral cancer and functioned as a potential factor that promoted the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells.Both ATRA and CD24 antibody might effectively inhibit the proliferation and tumorsphere formation of CAL-27 and WSU-HN6 cells and function as a potential therapy target.
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Objective To analyze the correlation between the expression of Abl interactor 1 (ABI1) and the clinicopathologic characteristics in colorectal adenocarcinoma tissues.Methods Immunohistochemistry was used to determine ABI1 expression in human colorectal adenocarcinoma tissues and matched adjacent tissues.A statistical analysis was used to determine the potential correlation between ABI1 expression and clinicopathological characteristics and prognosis.Results ABI1 is up-regulated in colorectal adenocarcinoma tissues compared with matched adjacent tissues (P =0.000),ABI1 expression is significantly correlated with infiltration (P =0.043) and differentiation (P =0.040),but not with sex,age,tumor location,clinical stage,lymph node metastasis and distant metastasis (P ＞0.05);It was also shown that ABI1 expression had a significant influence on prognosis (x2 =11.090,P =0.001).Multivariate analyses showed that high ABI1 expression is not an independent poor prognostic factor for overall survival (P =0.119).Conclusion ABI1 overexpression might act as pro-oncogene for patients with colorectal adenocarcinoma.
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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.
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A wireless electrochemical recording device was designed for in_vivo neurotransmitters real_time detection. Low_power microcontroller MSP430 was chosen as main control unit in hardware system. Other modules were current detection module, waveform generator module and data transceiver module. This device had the merits of small size (2. 3 cm×1. 8 cm×0. 6 cm) and low power consumption. Firmware program design was based on uC/OS operating system. Combined with the PC software, the device could achieve online display and analysis of the recording data. For neurotransmitter detecting needs, the device implemented fast_scan cyclic voltammetry ( FSCV) and fixed_potential amperometry. By using fast_scan cyclic voltammetry method, a linear relationship ( R=0. 99 ) between the concentration of dopamine and response current was acquired in the range of 5. 0×10-7-7. 0×10-5 mol/L. In the in_vivo experiments, the electrically evoked dopamine was recorded in the caudate_putamen area of brain in rats. Experimental results showed that the system had high detection accuracy, which could realize qualitative and quantitative analysis of the brain neurotransmitter. This work would have a broad application prospect in the field of neuroscience research.
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Purpose To study the clinicopathologic features, differential diagnosis, immunophenotype and treatment and prognosis of adult intratesticular rhabdomyosarcoma ( ITRMS) . Methods One case of adult ITRMS was analyzed by histology and immunohisto-chemistry, the date of treatment and prognosis through electronic medical record. The published relevant literatures were reviewed. Re-sults A 19-years old male patient was admitted to the hospital with a history of 2-months testicular painless enlargement. Testis ultra-sound examination and scrotal and abdominal CT revealed a mass in right testis. Macroscopic observation of the testis showed that a grey-white tumor. The tunica albuginea, epididymis and spermatic cord hadn’ t been invaded by the tumor. Microscopically, medium-size oval or round cells were arranged in sheets, and spindle-shaped, strap-like, large-circle cells were also presented. Few large cells had abundant granular eosinophilic cytoplasm with crossstriations under oil immersion objective. Immunohistochemically, the tumor cells were positive for vimentin, HHF35, desmin, MyoD1, Myogenin and h-Caldesmon protein, more than 50% neoplasm cells were positive. The rate of Ki-67 was 75%. Conclusions Adult ITRMS is rare. Histologically, embryonal rhabdomyosarcoma ( ERMS) is the most common type. They have highly invasive and poor prognosis. The accurate diagnosis depends on the combination of the histo-logic and immunohistochemical examination.
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<p><b>BACKGROUND</b>Abnormal neuronal differentiation plays an important role in central nervous system (CNS) development abnormalities such as Down syndrome (DS), a disorder that results directly from overexpression of genes in trisomic cells. Receptor-interacting protein 140 (RIP140) is significantly upregulated in DS brains, suggesting its involvement in DS CNS development abnormalities. However, the role of RIP140 in neuronal differentiation is still not clear. The current study aimed to investigate the effect of RIP140 overexpression on the differentiation of neuro-2a (N2a) neuroblastoma cells, in vitro.</p><p><b>METHODS</b>Stably RIP140-overexpressing N2a (N2a-RIP140) cells were used as a neurodevelopmental model, and were constructed by lipofection and overexpression validated by real-time polymerase chain reaction and Western blot. Retinoic acid (RA) was used to stimulate N2a differentiation. Combining the expression of Tuj1 at the mRNA and protein levels, the percentage of cells baring neurites, and the number of neurites per cell body was semi-quantified to determine the effect of RIP140 on differentiation of N2a cells. Furthermore, western blot and the ERK1/2 inhibitor U0126 were used to identify the specific signaling pathway by which RIP140 induces differentiation of N2a cells. Statistical significance of the differences between groups was determined by one-way analysis of variance followed by the Dunnett test.</p><p><b>RESULTS</b>Compared to untransfected N2a cells RIPl40 expression in N2a-RIP140 cells was remarkably upregulated at both the mRNA and protein levels. N2a-RIP140 cells had a significantly increased percentage of cells baring neurites, and numbers of neurites per cell, as compared to N2a cells, in the absence and presence of RA (P < 0.05). In addition, Tuj1, a neuronal biomarker, was strongly upregulated in N2a-RIP140 cells (P < 0.05) and phosphorylated ERK1/2 (p-ERK1/2) levels in N2a-RIP140 cells were dramatically increased, while differentiation was inhibited by the ERK1/2-specific inhibitor U0126.</p><p><b>CONCLUSIONS</b>RIP140 overexpression promotes N2a cell neuronal differentiation by activating the ERK1/2 pathway.</p>
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Humans , Blotting, Western , Cell Differentiation , Physiology , Cell Line , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Neurons , Cell Biology , Metabolism , Nuclear Receptor Co-Repressor 1 , Metabolism , Signal Transduction , PhysiologyABSTRACT
Objective To analyze the correlation between the expression of phosphoinositide-3 kinase,catalytic subunit beta (PIK3CB) and the clinicopathologic characteristics in gastric adenocarcinoma tissues.Methods Immunohistochemistry was used to determine PIK3CB expression in human gastric adenocarcinoma tissues and matched adjacent tissues.The correlation between PIK3CB expression and the patients' clinicopathological characteristics and prognosis was statistically analyzed.Results PIK3CB is highly expressed in gastric adenocarcinoma tissues (positive rate 63.8％) compared with matched adjacent tissues (positive rate 45.7％) (x2 =9.139,P =0.003).PIK3CB expression is significantly correlated with tumor size (x2 =8.677,P =0.003),infiltration (x2 =9.306,P =0.025),and clinical stage (x2 =8.242,P =0.041) ; Level of PIK3CB expression has a significant impact on prognosis (x2 =25.644,P =0.000).Multivariate analyses showed that high PIK3CB expression is an independent poor prognostic factor for overall survival (HR =15.785,95％ CI:1.943-7.805,P =0.000).Conclusions Upregulated PIK3CB is correlated with poor postoperative prognosis of gastric carcinoma.
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OBJECTIVE@#To study the reference values for differential cell counts in nasal lavage of normality in Nanjing.@*METHOD@#The total and differential cell counts were examined in nasal lavage from samples of a total of 300 healthy non-smoking adult volunteers.@*RESULT@#Nasal lavage succeeded in 281 subjects, the achievement ratio was 93.67%. The proportions of eosinophils 0.46 +/- 1.03, neutrophils were 4.46 +/- 9. 84, macrophages 0. 19 +/- 0.73 and lymphocytes 0.04 +/- 0. 16, respectively. There were no significant differences in the differential cell counts between male and female(P>0. 05) . The 95% ceiling percentile of eosinophils and neutrophils in nasal lavage are 2. 58 and 19.76 respectively.@*CONCLUSION@#The reference values were established for differential cell counts in nasal lavage of normality in Nanjing. We propose that these data be used in research on pathogenesis, diagnosis and treatment of the patients with nasal inflammation.
Subject(s)
Adolescent , Adult , Female , Humans , Male , Middle Aged , Young Adult , Cell Count , Eosinophils , Cell Biology , Lymphocytes , Cell Biology , Macrophages , Cell Biology , Nasal Lavage , Neutrophils , Cell Biology , Reference ValuesABSTRACT
Objective To determine the expression of G-protein coupled receptor 34 (GPR34) in hepatocellular carcinoma tissues.Methods Immunohistochemistry and tissue-array were used to determine GPR34 expression in human hepatocellular carcinoma tissues and matched adjacent tissues.A statistical analysis was performed to establish the potential correlation between GPR34 expression and the patients'clinicopathological characteristics,tumor progression,and prognosis.Results GPR34 is up-regulated in primary hepatocellular carcinoma tissues compared with matched adjacent tissues (P =0.003),and it was shown that GPR34 expression is significantly correlated with tumor size (P =0.024),small hepatocellular carcinoma(0.030) and infiltration(P =0.012),but not with sex,age,grade,chnical stage (all P ＞0.05) ; it was also shown that GPR34 expression had a significant influence on prognosis (X2 =5.617,P=0.018).Multivariate analyses showed that high GPR34 expression is an independent poor prognostic factor for overall survival (P =0.037).Conclusions The up-regulation of GPR34 acts as an potential prooncogene in the development and progression of hepatocellular carcinoma.GPR34 may be a useful diagnostic or prognostic molecular biomarker,and a potential target for therapeutic intervention.
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Objective To study the effect and mechanism of CKLF-like Marvel transmembrane domain containing 5 (CMTMS) on prostate cancer cell proliferation,migration and invasion.Methods The inhibitory effects of CMTM5 on the migration of DU145 cells were studied in vitro by wound healing assay.The expression of the cell signal pathway PI3K-AKT protein was detected by Western blot.The inhibition of tumor growth was also studied in transplanted prostate cancer nude mice model treated with CMTM5 adenovirus.The expression of CMTM5 and ki-67 in transplanted prostate cancer tissue of the nude mice model was analyzed immunohisochemistically.Prostate tumor volume in the nude mice model and the proliferation were measured two weeksafter.injection..Results Wound healing assay showed that over-expression of CMTM5 can inhibit the migration of DU145 cells.The expression of pAKT and NF-kB was significantly decreased after the overexpression of CMTM5.The tumor volume (573.39 ± 175.24) mm3,weight (0.55 ± 0.11) g and proliferation index of prostate in CMTM5 orthotopic injection nude mice model were significantly smaller and decreased than those in the control group (1482.50 ± 327.86) mm3 and (1.31 ± 0.29) g (P ＜ 0.05).Conclusions Both in vitro and in vivo experiments demonstrate that overexpression of CMTM5 could suppress prostate cancer cell proliferation,migration and invasion.The effect may be conducted by PI3K-AKT signaling pathway.
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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.
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ObjectiveTo investigate the effects of ABI-1 gene knockdown upon the proliferation and migration of human gastric cancer cell NCI-N87 in vitro. MethodsNCI-N87-ABI-I-ShRNA cell model was successfully constructed and validated by Real-time PCR and Western blot. The cellular morphous and skeleton, proliferative and migrative potents, and also AKT expression were compared between NCI-N87-ABI-1-ShRNA and its parents by immunofluorental staining, CCK-8 assay, transwell chamber and Western blotting.ResultsCCK-8 assay showed there was no significant difference in the proliferation rates at different time points between the NCI-N87-Vector and NCI-N87 cells while the proliferation rates at the time points of 36 and 48 hours of the NCI-N87-ABI-1-ShRNA were significantly lower than the NCI-N87( t =2. 85and 4. 166, P ＜ 0. 05 ). Transwell assay showed that migrated cell number were 66 ± 8, 65 ± 8 and 30 ± 4,respectively, and there was significant difference between the NCI-N87-ABI-1-ShRNA and NCI-N87 cells (t =9. 550,P ＜0. 05). Finally, ABI-1- knock-down altered the cellular morphoos and skeleton of 90％NCI-N87 cells and inhibited p-AKT expression.ConclusionABI-1 inhibits proliferation and migration of NCI-N87 cells in vitro probably by PI3K/AKT pathway.
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A new and effective method to produce transgenic animals was established. Without a surgical incision, the recombinant plasmid containing green fluorescence protein (GFP) cDNA was repeatedly injected into male mouse testis at multi-sites. After few weeks of the final injection, the injected male was mated with normal oestrus female to produce transgenic mice. The presence of the GFP cDNA in F1 transgenic individuals were detected by polymerase chain reaction and Southern blot hybridization, which showed that the transgenic rate of mouse F1 offspring was 41%. The transferred gene was integrated into the host genome and could be transmitted to its offspring. When the positive F1 individuals were mated with the wild type ICR mice, the F2 individuals had a transgenic rate of 37%. The results indicate that the high efficiency of gene transfer and the limited number of manipulations make the method suitable for creating a large number of transgenic animals, especially, for producing domestic animals.
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Objective: To clone a novel gene and explore its expression patterns in tissues and cells,so as to find its role in the process of encephalopathy in DS.Methods: On the base of our previous microarray's result together with the tissue type,we chose EST AI480014 to carry out RACE,then analyzed its expression profiles in liver,spleen,kidney,heart,brain by multi-tissues Northern blot,after that semi-quantitive RT-PCR was used to reexamine the expression profiles.Furthermore,we used ISH to find whether aim gene expressed in neuroglial cells cultured in vitro.Finally we performed semi-quantitive RT-PCR to explore whether it expressed differently between DS and normal.Results: We gained a 682 bp new cDNA fragment(DQ275636)which expressed in all the tissues examined and had no alternative splices in them.It expressed highly in brain especially in frontal lobe and hippocampus.According to the ISH result we convinced that it expressed in neuroglial cells.Using bioinformatics we mapped DQ275636 to chromosome 5q14.Conclusion: We have obtained a new gene fragment based on the(above) results.According to its expression character and tissue type,it can be suggested that this gene has a probable role in the process of encephalopathy in DS.
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Objective: To explore the influence factors of the degenerate oligonucleotide primered PCR(DOP-PCR). Methods: Genome DNA template from the mouse single oocyte or liver tissue were used to perform DOP-PCR. DOP-PCR was carried out with templates of different origin, different gradient dilution, with or without low melting point gel purified to wipe off the small fragment that might interfere with the following analysis, and then PCR of gene FTCD and CBS were carried out to evaluate the influence of these factors on the amplification efficiency and specificity. Results: Compared with genome DNA template from mouse liver, the template from single oocyte had the same efficiency and specificity but a minor yield and different gradient dilution of DNA template had no effect on the efficiency and specificity. Furthermore, there was a higher specificity in the low melting point gel-purified DOP-PCR product than in untreated ones. Conclusion: We have got a satisfactory result and increased specificity from DOP-PCR product purified with the low melting point gel. Single oocyte of mice could be used for further investigation of special genes detection by DOP-PCR and of an optimization in the yield of the products.
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Objective To study the effect of expression of RNA interference targeting protein kinase B(PKB) gene transfection mediated by nanoparticles(NP) on intimal hyperplasia(IH) in vein grafts of rats.Methods Nanoparticle PKB short hairpin RNA(shRNA) gene complex was prepared with PLGA and PVA.Autogenous vein graft model was established in 72 rats by transplanting internal jugular vein to carotid artery.The models were randomly divided into 3 groups:(1) PKB shRNA group: PKB-shRNA gene mediated by NP were transfected into the veins before anastomosis.(2) Empty vector group: the veins were transfected by empty vector mediated by NP.(3) Control group(no transfection).The grafted veins were harvested 3,7,14 and 28 days after the operation respectively.The mRNA and protein expression of PKB were determined by Northern blot and Western blot.IH was observed by HE and Verhoeff stain.The presence of apoptotic VSMC was detected by TUNEL stain.Results Compared to empty vector group and control group,PKB shRNA group had less expression of mRNA and protein of PKB gene(P