Small interfering RNA (siRNA) is the initiator of RNA interference and inhibits gene expression by targeted degradation of specific messenger RNA. siRNA-mediated gene regulation has high efficiency and specificity and exhibits great significance in the treatment of diseases. However, the naked or unmodified siRNA has poor stability, easy to degrade by nuclease, short half-life, and low intracellular delivery. As an emerging non-viral nucleic acid delivery system, ionizable lipid nanoparticles play an important role in improving the druggability of siRNA. At present, one siRNA drug based on ionizable lipid nanoparticles has been approved for the treatment of rare disease. This review introduces the research progress in ionizable lipid nanoparticles for siRNA delivery, focusing on the effect of each component of lipid nanoparticles on the efficiency of siRNA-mediated gene silencing, which provides new references for the studies on ionizable lipid nanocarriers for siRNA delivery.

OBJECTIVE An UPLC method in vitro was established for simultaneous determination of seven active components in Qianyang Yuyin prescription, including gallic acid, protocatechuic acid, loganin, stilbene glucoside, cyasterone, cinnamic acid and harpagoside.METHODS The analysis was performed on an Waters H-Class UPLC C18 column (2.1 mm×100 mm, 1.7 μm) eluted with a gradient mobile phase of acetonitrile-0.1% formic acid solution system at a flow rate of 0.3 mL/min.The detective wavelength was set at 250nm, the column temperature was maintained at 30℃ and the injection volume was 2 μL.The 7 main chemical compounds in Qianyang Yuyin prescription were determined by UPLC.RESULTS The linear relationship of the 7 chemical constituents in Qianyang Yuyin Prescription were good in their respective concentration ranges(r>0.997 5), The average recoveries were 95.8%~103.75%, the RSD of average recovery were 0.1%~1.0%, the RSD of precision were 0.10%~0.56%, the RSD of stability were 1.25%~9.04% and the RSD of reproducibility were 1.01%~3.24%.CONCLUSION The new established method in Qianyang Yuyin prescription was simple, accurate and reliable, which will supply instruction and reference to the internal Pharmacokinetics of Qianyang Yuyin prescription in the late works of this issue.

OBJECTIVETo investigate the specific antitumor immune response induced by idiotype protein (Id)-pulsed dendritic cells (DC) in vitro.

METHODSDC was generated from peripheral blood monocytes of the multiple myeloma (MM) patients using GM-CSF, IL-4, and TNF-alpha. The DCs were pulsed with idiotypic fragment, the F(ab')2 fragment of M protein from MM patient at the immature stage. The morphologic characteristics of the cells were observed with light and electron microscopes. The phenotypic features were analyzed with FACS, MTT assay was employed to evaluate the proliferation of autologous T cells and the inhibition rate of MM cells.

RESULTSDC precursors in peripheral blood could be induced to typical mature DC in medium containing GM-CSF, IL-4 and TNF-alpha. Mature DC with Id could increase the proliferation of the autologous T cells and activate naive T cells to become tumor specialized cytotoxic T lymphocytes (CTL). The CTL at different doses showed significant inhibition on or killing ability to autologous MM cells in vitro.

CONCLUSIONSIn a suitable cytokine environment, the DC precursors from peripheral blood of MM patients could be induced to functional DC, and vaccination of Id-pulsed DC could induce active antitumor immune response.

Adult ; Aged ; Antibodies, Anti-Idiotypic ; immunology ; Cancer Vaccines ; immunology ; Cells, Cultured ; Dendritic Cells ; immunology ; Female ; Humans ; Immunotherapy, Active ; Male ; Middle Aged ; Multiple Myeloma ; immunology ; therapy ; T-Lymphocytes, Cytotoxic ; immunology

Objective A SYBR-Green real-time quantitative PCR(RT-qPCR)method was set up to detect the infection and proliferation of measles virus, which could be useful in virus titer determination. Methods In this project, we used a 405 bp fragment of the N gene of measles virus as a target sequence and constructed a plasmid to establish the standard curve in absolute quantitative experiment. We then used this method to obtain the proliferation curve of measles virus and to detect the virus proliferation at different MOI. Results There was a linear relationship between the virus copy number and the titer of the measles virus reference at the range of 6 to 2 lgCCID₅₀/mL, with a correlation coefficient (r) of 0.991(P < 0.01). Based on the analysis of virus proliferation curve, measles virus mainly proliferated intracellularly within 48 h after its entering the cell. There was no detected increase in viral RNA level in the first 24 h, suggesting the virus was in a silent period in the cell. After 24 h, the virus expanded in large numbers and entered the exponential growth phase. The intracellular viral RNA level reached the plateau phase after its peak at 96 h. The virus secreted to the outside of the cell entered the exponential growth phase starting from 48 h, peaked at 144 h, then followed by plateau phase. Conclusion A SYBR-Green RT-qPCR method is established and used to monitor virus proliferation. Our result is helpful in understanding of the proliferation and secretion of measles virus in cells and provides experimental basis for detection of live attenuated virus titers.

Objective To analyze the genetic diversity of Pomacea canaliculata based on the mitochondria DNA cytochrome c oxidase subunitⅠ(mtDNA COⅠ)gene as a molecular marker in Lincang City of Yunnan Province,so as to provide the scien-tific data for monitoring Angiostrongylus cantonensis in local areas. Methods The genotypes and polymorphisms of 38 speci-mens of P.canaliculata collected from Mengding Town of Lincang City were analyzed by sequencing COⅠgene.The phylogenet-ic tree and genetic distances were produced based on the haplotypes from GenBank and the present study by using the neighbour-joining method with the software MEGA version 6.06. Results Totally 31 sequences were acquired in the present study and they produced 3 unique haplotypes.Haplotype 1 showed a higher frequency compared to the others and it accounted for 83.9 % (26/31).The data showed that the least genetic distances ranged from 0 to 0.052 between P.canaliculata and 3 haplotypes,as well as the largest genetic distances ranged from 0.021 to 0.239 between Pila conica and 3 haplotypes.Otherwise,the analysis of the phylogenetic trees based on COⅠgene sequences of P.canaliculata indicated that all of 3 haplotypes clustered into one big clade with that from Japan(GenBank accession number: AB433769),China(GenBank accession number: KT313034)and USA(GenBank accession number:EU523129),which owned the closet relationship amongst them.Their genetic relationships were distantly related to the GenBank's reference sequences of P.insularum(GenBank accession number:EF514942),P.cam-ena(GenBank accession number: EF515059)and so on. Conclusion There is a P. canaliculata species in Lincang City of Yunnan Province as well as a high genetic diversity amongst the acquired 3 haplotypes in this study.

OBJECTIVETo investigate the expression of transcription factor Sp1 in human gastric cancer tissues and normal gastric mucosa, and its prognostic significance.

METHODSBy using immunohistochemistry, we studied the Sp1 expression patterns in 65 cases of gastric cancer with various clinico-pathologic characteristics, and 40 normal gastric mucosa specimens obtained from patients who underwent partial gastrectomy for benign gastric diseases. The significance of Sp1 expression on the survival of patients was evaluated.

RESULTSThe expression rate of Sp1 in normal gastric mucosa was 12.5% (5/40). The positively stained glandular cells were mainly limited to those in the neck region. Cells at the basal portion of the gland were essentially negative. In sharp contrast, Sp1 expression rate in gastric cancer lesions was 53.8% (35/65). The medium survival time in patients who had a tumor with negative, weak and strong Sp1 expression was 1700, 1560 and 1026 days, respectively (P = 0.036). Sp1 protein expression was closely related to the depth of tumor invasion and TNM stage (P = 0.001, P = 0.026), but not related to the number of metastatic lymph nodes and Lauren's classification (P = 0.306, P = 0.667).

CONCLUSIONNormal and malignant gastric tissues have unique Sp1 expression patterns. Sp1 might be served as an independent prognostic factor.

Adenocarcinoma ; metabolism ; pathology ; Aged ; Biomarkers, Tumor ; biosynthesis ; genetics ; Female ; Follow-Up Studies ; Gastric Mucosa ; metabolism ; Humans ; Male ; Middle Aged ; Neoplasm Invasiveness ; Prognosis ; Sp1 Transcription Factor ; biosynthesis ; genetics ; Stomach Neoplasms ; metabolism ; pathology

OBJECTIVETo investigate the expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor-C (VEGF-C) in human gastric cancer, the relationship between their expression and the clinicopathological features,as well as the relationship between these two parameter expression and lymphangiogenesis and lymph node metastasis.

METHODSCOX-2 and VEGF-C expressions were detected in 63 gastric cancer samples by immunostaining. Lymphangiogenesis was evaluated by immunostaining with the specific antibody LYVE-1.

RESULTSThe expression rates of COX-2 and VEGF-C were 66.7% (42/63), 52.4% (33/63), respectively in 63 gastric cancer specimens. LYVE-1 was positive in 35 cases (35/63), which indicated lymphangiogenesis in the tumors. The expression of COX-2 was significantly correlated with the expression of VEGF-C, tumor lymphangiogenesis and lymphatic metastasis (P< 0.05), however not gender, tumor size, tumor location, Lauren classification and serosa invasion (P< 0.05).

CONCLUSIONSIn gastric cancer, the expression of COX-2 is significantly associated with VEGF-C expression, lymphangiogenesis and lymphatic metastasis. COX-2 may up-regulate the expression of VEGF-C, which induces lymphangiogenesis and accordingly contributes to lymphatic metastasis.

Aged ; Cyclooxygenase 2 ; metabolism ; Female ; Humans ; Lymph Nodes ; metabolism ; pathology ; Lymphangiogenesis ; Lymphatic Metastasis ; Male ; Middle Aged ; Stomach Neoplasms ; metabolism ; pathology ; Vascular Endothelial Growth Factor C ; metabolism

OBJECTIVETo study the clinical value of endoscopic ultrasonography (EUS) in the preoperative staging of early gastric carcinoma.

METHODSEUS was performed in 149 gastric carcinoma patients proved by biopsy (including 33 patients with early gastric cancer), of which the results were compared with postoperative pathologic findings.

RESULTSThe accuracy of EUS in determining the T stage of gastric carcinoma was 80.3% (T1 81.8%, T2 70.4%, T3 88.9%, T4 71.4%). The accuracy of EUS in differentiating early gastric carcinoma from advanced ones was 95.1%, and the accuracy of EUS in differentiating mucosal cancer from submucosal cancer was only 63.6%. The diagnostic accuracy of EUS for mucosal and submucosal cancer was 52.9% and 75%, with positive predictive value of 90% and 70.6%, respectively. The accuracy of invasion depth of EUS for the bulging and flat type of early gastric carcinoma was 100%, whereas the accuracy was only 58.6% for the depressed type. The accuracy of invasion depth of the differentiated and undifferentiated early cancer was 71.4%and 57.9%, without any significant difference (P > 0.05). The accuracy of invasion depth of EUS for early gastric carcinoma decreased as tumor size increased. The diagnostic accuracy of lymph node status of early gastric carcinoma by EUS was 90.9%, and the sensitivity and specificity of lymph node metastasis was 66.7% and 96.3%, respectively.

CONCLUSIONThe clinical value of endoscopic ultrasonography in the preoperative staging of early gastric carcinoma is relatively high.

Adenocarcinoma ; diagnostic imaging ; pathology ; surgery ; Adult ; Aged ; Biopsy ; Endosonography ; Female ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; Preoperative Care ; Stomach ; pathology ; Stomach Neoplasms ; diagnostic imaging ; pathology ; surgery

OBJECTIVETo determine the microsatellite instability in gastric carcinomas, examine the frameshift mutations of transforming growth factor-beta type II receptor (TGFbetaRII), insulin growth factor II receptor (IGFIIR), bcl-2 associated X protein (BAX) and E2F4, and investigate whether or how alterations of the TGFbetaRII, IGFIIR, BAX and E2F4 gene are associated with MSI in gastric cancer.

METHODSFormalin-fixed, paraffin-embedded gastrectomy specimens and matching normal tissues of 65 cases of gastric carcinomas were retrieved from shanghai Ruijin Hospital and Shanghai East Hospital. DNA was extracted from tissue sections using phenol chloral isoamyl alcohol. Sections with no more than 50% of tumor cell areas were isolated by microdissection. DNA was amplified by PCR-based single strand conformation polymorphism (SSCP) for microsatellite analysis and was sequenced directly. Frameshift mutations in the coding regions, repetitive mononucleotide tracts of (A)10 for TGFbetaRII, (G)8 for IGFIIR, (G)8 for BAX, and trinucleotide repeats of (AGC)13 for transcription factors E2F4 were detected using PCR. Tumors were classified as being microsatellite stable (MSS) or having a low frequency of MSI (MSI-L, one of markers different in the tumor) or a high frequency of MSI (MSI-H, two or more of markers different).

RESULTSEleven cases (18.0%) showed MSI-L, 12 (19.7%) showed MSI-H and 38 (62.3%) cases showed MSS. The mutation rates of TGFbetaRII, IGFIIR, BAX and E2F4 gene were 19.7%, 4.9%, 6.6% and 16.4% respectively. Among the 12 MSI-H gastric cancers, there were 10 TGFbetaRII mutations, 3 IGFIIR mutations, 4 BAX mutations and 10 E2F4 gene mutations. The alterations in the repeats of the related genes presented polymorphisms. Associations of MSI-H status and mutations of the 4 genes were highly significant (P < 0.01, respectively). No repeat tracts mutations in TGFbetaRII, IGFIIR, BAX and E2F4 gene were found in MSS tumors.

CONCLUSIONSThe repeat coding regions within TGFbetaRII, IGFIIR, BAX and E2F4 gene are the targets of microsatellite instability. Frameshift mutations of the 4 genes play an important role in the development and progression of gastric cancers with microsatellite instability.

Adult ; Aged ; Aged, 80 and over ; China ; E2F4 Transcription Factor ; genetics ; Female ; Frameshift Mutation ; Humans ; Male ; Microsatellite Instability ; Middle Aged ; Polymerase Chain Reaction ; Protein-Serine-Threonine Kinases ; Receptor, IGF Type 2 ; genetics ; Receptors, Transforming Growth Factor beta ; genetics ; Stomach Neoplasms ; genetics ; bcl-2-Associated X Protein ; genetics

OBJECTIVETo investigate the value of transabdominal ultrasonography (TAUS) in preoperative assessment of TNM stage and tumor angiogenesis for patients with gastric carcinoma.

METHODSSixty- four patients with gastric carcinoma preoperatively underwent TAUS, in whom transabdominal color Doppler ultrasonography was used for measuring color Doppler vascularity index (CDVI) of each tumor in 37 cases and microvessel density (MVD) was evaluated by using immunohistochemical staining of surgical specimens with anti- CD34 antibody.

RESULTSThe overall accuracy rate was 56.0% for T staging of gastric carcinoma (T (1) 2/3 cases, T (2) 28.6% , T (3) 73.1% , T (4) 50.0% , respectively) by TAUS. The diagnostic accuracy rate was 63.3% for lymph node status of gastric carcinoma. The diagnostic sensitivity and specificity for lymph node metastasis was 37.9% and 100% respectively. The overall accuracy for N staging of gastric carcinoma was 57.1% (N (0) 100% , N (1) 16.7% , N (2) 35.3% , respectively). The diagnostic sensitivity and specificity for determining distant metastases was 58.3% and 100% respectively. The CDVI of gastric carcinoma determined by color Doppler ultrasonography was significantly correlated to vascular invasion (P=0.0418), a linear correlation between CDVI and MVD was determined by logistic regression analysis (r=0.5628, P< 0.01).

CONCLUSIONTAUS can be a routine diagnostic approach for preoperative gastric carcinoma patients.

Abdomen ; diagnostic imaging ; Adult ; Aged ; Female ; Humans ; Male ; Microvessels ; Middle Aged ; Neoplasm Staging ; Neovascularization, Pathologic ; diagnostic imaging ; Stomach Neoplasms ; diagnostic imaging ; pathology ; Ultrasonography, Doppler, Color ; methods