Objective:To investigate the expression of RAD18 in colon cancer and its correlation with proliferating cell nuclear antigen (PCNA).Methods:The glass slice of colon cancer tissues and adjacent normal tissues from patients (73 cases) who underwent surgical treatment at the Second Hospital of Tianjin Medical University from November 2013 to November 2023 were collected.The expression of RAD18 in colon cancer tissues and adjacent normal tissues was analyzed in the gene expression profiling interactive analysis (GEPIA) database and verified by immunohistochemical staining. The relationship between RAD18 expression and clinicopathological features of colon cancer patients was analyzed. HCT116 and HT29 cells were cultured in vitro, and the control group and transfection group ( transfected with RAD18 shRNA to knock down RAD18 ) were set up. The expression of RAD18 was evaluated by quantitative real-time PCR (qRT-PCR) and Western Blot. The effect of RAD18 on colon cancer cell proliferation was explored using clonogenic assays and cell counting Kit-8 (CCK-8) assays. The correlation between RAD18 and PCNA was investigated by GEPIA and immunohistochemical staining. Results:The GEPIA database analysis showed that the expression of RAD18 in colon cancer tissue ( n = 275) was significantly higher than that in adjacent normal tissues ( n = 349, P < 0.05). RAD18 was expressed at higher levels in colon cancer tissue than that in adjacent normal tissues and was not expressed at high levels in the latter. The expression of RAD18 was closely related to tumor size in the low-expression group and high-expression group of patients ( P = 0.015) but was not related to age ( P = 0.115), gender ( P = 0.665), or tumor differentiation ( P = 0.733). Compared with the control group, the expressions of RAD18 in the transfection group of HCT116 and HT29 cells were both reduced (both P < 0.05). Compared with the control group, the clone cell number and absorbance ( A) value of HCT116 and HT29 cells in the transfection group were decreased (all P < 0.05). GEPIA database analysis showed that RAD18 was correlated with PCNA ( R = 0.27, P < 0.05), and the expression level of PCNA was higher in colon cancer tissues than in adjacent normal tissues. Conclusions:RAD18 is expressed at a higher level in colon cancer tissues and may promote colorectal cancer proliferation by affecting PCNA.

Objective:To explore the molecular mechanism of Matrine Injection in treating colorectal cancer based on network pharmacological analysis and molecular docking.Methods:Taking matrine as the object, the corresponding potential drug targets in matrine were obtained from Swiss Target Prediction database, and SuperPred database, and the database of traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP). Differential genes were obtained from gene expression omnibus (GEO), and GeneCards, OMIM, DrugBank, and CTD databases were used to collect colorectal cancer-related genes. Furthermore, core targets were screened by establishing protein-protein interaction (PPI) networks. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were performed by bioconductor of R language. Finally, the molecular docking calculation was performed to evaluate the interaction between matrine and core targets.Results:Matrine contained 63 targets. A total of 14 198 targets for colorectal cancer were obtained. The topology analysis results of the PPI network showed that 5 main targets such as myelocytomatosis proteins (MYC), interleukin-6 (IL-6), Caspase-3 (CASP3), mammalian target of rapamycin (mTOR), and amphiregulin (AR). GO enrichment analysis found that biological process (BP) mainly includes hydrogen peroxide reaction, cell reaction to hydrogen peroxide and cell response to chemical stress, etc; Cell components (CC) mainly include lipid rafts, membrane microregions and synaptic membranes, etc; Molecular functions (MF) mainly include transcriptional coregulatory factor binding, postsynaptic neurotransmitter receptor activity and core promoter sequence-specific DNA binding. KEGG pathway analysis showed that it involved chemical carcinogenesis-receptor activation, tumor necrosis factor (TNF) signaling pathway, and Toll-like receptor signaling pathway, etc. Molecular docking showed that matrine had good binding with the core target.Conclusions:Matrine acts on targets such as MYC, IL-6, CASP3, mTOR, and AR, and exerts therapeutic effects on colorectal cancer by regulating chemical carcinogenesis-receptor activation, TNF signaling pathway, Toll-like receptor signaling, etc.

Saccharomyces boulardii is a subspecies of Saccharomyces cerevisiae and is a fungal probiotic. It can regulate the intestinal flora and enhance the barrier function of the intestinal tract. Compared with bacterial probiotics, Saccharomyces boulardii is more resistant to acid and oxidation, does not transmit genetic material with bacteria, and can be used in combination with antibiotics. Saccharomyces boulardii can function through a variety of mechanisms, and many proteases secreted by it have antitoxin effects; its own bacteria contain more polyamines, which can nourish the intestinal mucosal cells and regulate the body's metabolic balance. Besides, it can regulate multiple signal pathways to enhance intestinal immunity. Saccharomyces boulardii has been used in the treatment of ulcerative colitis (UC). The results of animal experiments and clinical studies have shown that the application of Saccharomyces boulardii can improve intestinal inflammation and enhance the therapeutic effect of mesalazine. Saccharomyces boulardii can be used as an auxiliary drug for the treatment of UC.

Objective:To investigate the expression and clinical significance of thymidine kinase 1 (TK1), carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, CA15-3 and CA72-4 in colorectal tumors.Methods:Enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence immuno assay (ECLIA) were used to determine the serum TK1 levels and serum CEA, CA19-9, CA15-3, CA72-4 levels in 124 patients with colorectal cancer, 52 patients with colorectal precancerous lesions, 154 patients with benign colorectal lesions, and 106 health subjects. The relationship between serum TK1 and its clinicopathological characteristics in patients with colorectal cancer were analyzed. The diagnostic efficacy of TK1, CEA, CA19-9, CA15-3 and CA72-4 alone and combined detection for colorectal cancer was investigated.Results:The serum expression level of TK1 in patients with colorectal cancer was related to tumor stage, degree of differentiation, lymph node metastasis, distant metastasis and age (all P<0.05), but not related to the patient's gender ( P>0.05). The serum TK1 level decreased sequentially in colorectal cancer patients, precancerous lesions patients, benign lesions patients and healthy subjects. Colorectal cancer patients with high TK1 expression have a shorter survival time. The sensitivity, specificity and accuracy of the combined detection of TK1, CEA, CA19-9, CA15-3 and CA72-4 were 93.5%, 93.0%, and 93.1%, respectively. Conclusions:Serum TK1 is expected to become an independent marker for the diagnosis and prognosis of colorectal cancer. The combined detection of TK1, CEA, CA19-9, CA15-3 and CA72-4 has clinical significance in the diagnosis of colorectal cancer.

The incidence of colorectal cancer is high threatening human health. About 60％～70％cases of CRC are derived from colorectal polyps, which can be treated by endoscopic electrotomy to prevent the possibility of canceration. Therefore, in the prevention and treatment of CRC, the role of screening is of great significance. CRC screening methods include the most commonly used fecal occult blood test ( FOBT ) and the more sensitive fecal immunochemical test (FIT), cost-effective fiber sigmoidoscopy and colonoscopy, CT colonoscopy (CTC), and fecal DNA testing and immature CRC hematology screening. In this paper, the CRC screening technologies were reviewed, including the principles, characteristics and the latest research progress to provide a theoretical basis for the application and development of CRC screening technology.

Aim To investigate the effects of classic calcium antagonists verapamil(Ver),nifedipine(Nif),diltiazem(Dil)and the novel calcium antagonist N-n-butyl haloperidol iodide(F2)which was synthesized by our lab by regulating Ca2+-independent phospholipase A2(iPLA2)on hypoxia/reoxygenation(H/R)injury of cardiac microvascular endothelial cells(CMECs)and the mechanisms.Methods The CMECs were isolated from SD neonatal rats.The H/R model was established,then cells were treated with different concentrations of calcium antagonists and F2.The content of LDH in the cell supernatant was measured by colorimetric method.The levels of IL-6 and AA in cell supernatant were measured by ELISA;and late-stage apoptosis was measured by TUNEL.The mRNA and protein expression levels of iPLA2 in CMECs were examined by real time-PCR and Western blot analysis.Results Calcium antagonists except Dil decreased the generation of LDH,IL-6 and AA in a dose-dependent manner(P<0.05),and reduced the apoptosis(P<0.05).F2 and Ver decreased the mRNA and protein expression of iPLA2 in a dose-dependent manner,while there were no such effects for Nif and Dil.Conclusions Calcium antagonists except Dil have protective effects against H/R injury.F2 and Ver protect CMECs against H/R injury partly through iPLA2.

Aim To investigate the effect of N-n-butyl haloperidol iodide(F2) on mitochondria-dependent apoptotic pathway of H9c2 cardiac myocytes during hypoxia/reoxygenation(H/R) injury.Methods The H/R models of H9c2 cardiac myocytes were established.The H9c2 cardiac myocytes were randomly divided into five groups: control group(C group), hypoxia/reoxygenation group(H/R group), F2 low concentration group(L), F2 medium concentration group(M), F2 high concentration group(H).Apoptotic rate was evaluated by flow cytometry(FCM).The levels of Cyto C, Bcl-2, Bax were observed by Western blot.Caspase-3 activity was measured with colorimetry.Results Compared with H/R group, F2 low, medium and high concentrations group could significantly decrease apoptosis rate and increase the ratio of Bcl-2 to Bax proteins and inhibit the release of Cyto C into the cytosolic fraction, and decrease caspase-3 activity.Conclusion F2 can protect H9c2 cardiac myocytes against H/R-induced injury through interfering in mitochondria-dependent pathway.

OBJECTIVETo compare the clinical effect of graciloplasty using two different gracilis encircled loops and to research the better method for the treatment of fecal incontinence after anoplasty for imperforate anus.

METHODSClinical data of 38 incontinence patients with congenital anal malformation undergoing graciloplasty in our hospitals from January 2009 to January 2012 were retrospectively analyzed. Twenty patients received the modified surgery in which the gracilis muscle was transposed anticlockwise into a circum-anal tunnel with a U-loop and its tendon secured to the ipsilateral pectineal ligament. Eighteen patients received the traditional surgery in which the gracilis muscle was wrapped clockwise around the anus with a γ-loop and its tendon secured to the contralateral periosteum of ischial tuberosity or pectineal ligament. All the patients were evaluated via Wexner score and anal manometry before surgery and 2 weeks, 6 months, 1 year and 2 years after surgery. In addition, it was assessed whether the patients had difficulty defecating while squatting after surgery.

RESULTSGeneralized estimating equations showed that the Wexner score in two groups gradually decreased after surgery (P=0.000), but there was no significant difference between two groups (P=0.554). At 2 weeks, 6 months, 1 year and 2 years after surgery, there were respectively 4 cases (20.0%), 3 cases (15.0%), 2 cases (10.0%), 1 case (5.0%), who showed squatting difficult defecation in the U-loop group, and 10 cases (55.6%), 12 cases (66.7%), 10 cases (55.6%), 8 cases (44.4%) in the γ-loop group. The probability of squatting difficult defecation in U-loop group was significantly lower compared to γ-loop group (P=0.002), but the probability of squatting difficult defecation in two groups did not obviously change with time (P=0.057). Repeated measures ANOVA showed that anal resting pressure, anal maximal squeeze pressure, duration of anal squeeze, and rectal maximum tolerable volume in two groups were gradually improved after surgery (all P<0.01), but there were no significant differences between two groups (all P>0.05).

CONCLUSIONSGraciloplasty with different gracilis loops can improve anal function. However, U-loop can better improve difficult defecating while squatting, and is worthwhile for spreading in clinical practice.

OBJECTIVETo explore the clinical significance of vasohibin-1 expression in colorectal cancer tissues and its correlation with vascular endothelial growth factor A(VEGF-A) and microvessel density (MVD).

METHODSTumor tissues and paired adjacent normal tissue (distance to cancer >5 cm) from 60 colorectal cancer patients undergoing resection in the Second Hospital of Tianjin Medical University from June 2013 to November 2013 were included in this study. The protein expressions of vasohibin-1, VEGF-A and MVD were detected by immunohistochemical staining. The mRNA expressions of vasohibin-1 and VEGF-A were detected by RT-PCR. The protein expressions of vasohibin-1 and VEGF-A were observed by Western blot. Correlation among parameters was examined.

RESULTSVasohibin-1 expression was mainly localized in the cytoplasm of tumor cells and endothelial cells. VEGF-A expression was mainly localized in the cytoplasm and membrane of tumor cells. The expressions of vasohibin-1, VEGF-A and MVD in colorectal tumor tissues were significantly higher than those in corresponding adjacent tissues [43.3% (26/60) vs. 16.7% (10/60), 51.7%(31/60) vs. 18.3% (11/60), (39.67 ± 16.80)/mm² vs. (17.85 ± 6.43)/mm², all P<0.05]. Higher vasohibin-1 expression was significantly associated with TNM stage and metastasis (P<0.05). Vasohibin-1 expression was positively correlated with VEGF-A and MVD (r=0.378, 0.628, all P<0.05). Vasohibin-1 and VEGF-A mRNA expressions and protein expressions in colorectal cancer tissues were significantly higher than those in corresponding adjacent tissues (all P<0.05).

CONCLUSIONVasohibin-1 expression in colorectal cancer tissues is significantly higher as compared to corresponding adjacent tissues. Vasohibin-1 expression is positively correlated to VEGF-A and MVD, and associated to TNM stage and metastasis. Positive vasohibin-1 expression indicates a poor prognosis of patients with colorectal cancer.

Ranpirnase (onconase, ONC) is a new drug, with weak RNase activity and strong cytotoxicity to various tumor cells in vitro and in vivo. This study is to obtain recombination onconase (rONC) with high bioactivity. Based on the codon preference of Pichia pastoris, we designed and synthesized the gene according to cDNA sequences of ONC and the α mating factor's prepeptide. We screened positive clones after transforming the recombination plasmids into P. pastoris X-33, GSS115 and SMD1168. We screened the best combination of seven different vectors and host strains. Moreover, we optimized culture condition in shake flasks and 10 L bioreactor, and purified rONC from the supernatant after inducing it with 0.25% methanol by aqueous two-phase extraction coupling G50 molecular exclusion method. The highest rONC production was 13 mg/L in pPICZα-A/X-33/ONC combination under the condition of pH 5.5 and 23 degrees C in shake flasks for 7 d; and that the highest rONC production was 180 mg/L when the induction is performed in the lower basic salt medium with pH 5.5 in the 10 L bioreactor for 7 d. The yield of rONC is more than 90% at a purity of above 95%. rONC can kill various tumor cells in vitro. The expression and purification of rONC would be useful for further investigation of this new drug.
Antineoplastic Agents ; metabolism ; Bioreactors ; Cell Line, Tumor ; Codon ; DNA, Complementary ; Genetic Vectors ; Humans ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; Ribonucleases ; biosynthesis