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Objective:To analyze the diagnostic value of metabolic makers in cerebrospinal fluid in advanced lung adenocarcinoma patients with leptomeningeal metastases (LM) .Methods:A total of 46 cerebrospinal fluid samples (LM group) from lung adenocarcinoma patients with LM admitted to Nanjing Drum Tower Hospital Affiliated to Nanjing University Medical School from December 2019 to December 2021 were collected, and 48 cerebrospinal fluid samples (control group) from patients with benign neurological diseases during the same period were collected. Metabolomics analysis of cerebrospinal fluid was carried out by high-performance liquid chromatography-mass spectrometry. Principle component analysis (PCA) and orthogonal to partial least squares discriminant analysis (OPLS-DA) were used for modeling. Multi-criteria assessment was used to identify the different metabolites between the two groups. Receiver operating characteristic (ROC) curve, pathway enrichment analysis and other methods were used to screen metabolites and pathways related to LM from lung adenocarcinoma.Results:There were no statistically significant differences in the proportions of age ( Z=-0.41, P=0.210) , gender ( χ2=1.19, P=0.275) , history of smoking ( χ2=2.86, P=0.091) , Karnofsky performance status score ( χ2=0.65, P=0.419) and increased intracranial pressure ( χ2=0.65, P=0.419) between the LM group and control group. The models of PCA (R2X was 0.608 and 0.583, Q2 was 0.462 and 0.513 in electrospray ion positive and negative modes, respectively) and OPLS-DA (R2Y was 0.967 and 0.889, Q2 was 0.959 and 0.852 in electrospray ion positive and negative modes, respectively) showed that the overall data quality was good. Meanwhile, the model interpretation rate and prediction rate were effective. The permutation tests duplicated for 200 times and showed no over-fitting of the established model. The metabolic profiles of the two groups were significantly different. A total of 30 endogenously differential metabolites were screened by using multi-criteria assessment. Six potential biomarkers with larger area under the curve (AUC) were identified through ROC curve analysis, including tyrosine (AUC=0.967, 95% CI: 0.906-1.000) , phenylalanine (AUC=0.992, 95% CI: 0.973-1.000) , pyruvate (AUC=0.976, 95% CI: 0.935-1.000) , tryptophan (AUC=0.935, 95% CI: 0.880-0.973) , glucose (AUC=0.932, 95% CI: 0.880-0.975) and adenosine monophosphate (AUC=0.993, 95% CI: 0.987-1.000) . The 30 selected differential metabolites were enriched and analyzed for metabolic pathways, and 20 relevant metabolic pathways were matched. Among them, the four metabolic pathways most likely to cause changes in metabolites were glycolysis and glucose metabolic synthesis, pyruvate metabolism, phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis. Conclusion:Untargeted metabolomics analysis can effectively screen specific cerebrospinal fluid metabolites in lung adenocarcinoma patients with LM. Six potential metabolites such as tyrosine, phenylalanine, pyruvate, tryptophan, adenosine monophosphate, glucose and their metabolic pathways may be involved in the pathogenesis of LM from lung adenocarcinoma.
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BACKGROUND@#Tumor markers (TM) in cerebrospinal fluid (CSF) are useful for diagnosing leptomeningeal metastasis (LM). It has not been fully exploited the diagnostic possibilities of the CSF levels since the basic fact that the TM concentration of CSF depends strongly upon the serum levels as well as upon the condition of the blood brain barrier (BBB). To analyze the intrathecal TM synthesis and evaluate the integrity of BBB can be helpful for the definitive diagnosis of LM. Therefore, the aim of this study was to further explore the clinical value of intrathecal TM synthesis and BBB in the diagnosis for the lung cancer patients with LM.@*METHODS@#Twenty-five lung cancer patients with LM and 57 patients with nonmalignant neurological diseases (NMNDs) admitted to Nanjing Drum Tower Hospital from December 2016 to March 2020 were included. We compared the integrity of BBB and intrathecal TM synthesis between two groups, analyzed the correlation of CSF TM between the detection and intrathecal synthesis, and evaluated serial CSF cytology, the integrity of BBB and intrathecal TM synthesis when intrathecal chemotherapy for one patient.@*RESULTS@#Ninety-four percent LM patients showed the dysfunction of BBB, and all LM patients showed at least one intrathecal synthesized TM in CSF. In one patient, the CSF cytology was negative for the first time, but LM was eventually diagnosed based on the the intrathecal TM synthesis and positive CSF cytology of repeated lumbar puncture. In LM group, no correlation was observed between the detection and intrathecal synthesized TM in CSF. In the control group, only 3.5% (2/57) NMNDs patients had the dysfunction of BBB and no patients had intrathecal TM synthesis, both the differences of which were statistically significant (P<0.05). Finally, evaluating the CSF cytology, integrity of BBB and intrathecal TM synthesis can be used to assess the intracranial treatment effect. Moreover, intrathecal TM synthesis changes earlier than cytology.@*CONCLUSIONS@#The evaluation of intrathecal TM synthesis and integrity of BBB are novel clinical diagnostic tools. In addition, serial measurement of intrathecal synthesized TM may play an important role in monitoring efficacy of lung cancer patients with LM, which is worthy of further promotion and clinical application.
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BACKGROUND@#Leptomeningeal metastasis (LM) is one of the most common causes of death in patients with advanced non-small cell lung cancer (NSCLC), which is defined as malignant cells spreading to meninges and cerebrospinal fluid (CSF). Therefore, early diagnosis and timely treatment are essential. CSF cytology is the gold standard for LM diagnosis, however, it has a low sensitivity for diagnosis and can't be used to evaluate the treatment effect. The aim of this study was to assess the clinical value of serum and CSF tumor markers (TM) in the diagnosis and treatment of NSCLC patients with LM.@*METHODS@#Nineteen patients with NSCLC-LM and 27 patients with nonmalignant neurological diseases (NMNDs) were included. We tested the levels and positive rates of carbohydrate antigen (CEA), carbohydrate antigen-125 (CA125), cytokeratin 19 fragments (CYFRA21-1) and neurone specific enolase (NSE) in CSF and serum, compared the sensitivity and specificity in the diagnosis of LM between different groups, and analyzed the correlation of detection between serum and CSF. Finally, we measured serum and CSF TM dynamically in 2 patients with NSCLC developing LM in an attempt to correlate these with the treatment response of extracranial and intracranial, respectively.@*RESULTS@#The levels and positive rates of TM in CSF and serum in LM group were higher than those in NMNDs (P0.05). In CSF, CYFRA21-1 has the highest sensitivity (88.2%) and CEA has the best specificity (92.3%) to distinguish patients between LM and NMNDs. For combined detection of CEA, CA125, CYFRA 21-1 and NSE in CSF, when at least CEA or NSE was positive in patients with LM, the sensitivity and negative predictive value were 100.0%, and the specificity was 74.1%. When both CYFRA21-1 and NSE were positive, the specificity and positive predictive value were 100.0%, and the sensitivity was 78.9%. Furthermore, subgroup analysis showed that the detection rates of TM in CSF cytology positive population was higher than that in typical abnormalities magnetic resonance imaging population, but there was no statistical difference (P>0.05). The detection of TM between serum and CSF in LM patients had no significant correlation. Moreover, biochemical properties of CSF from ventricle and lumbar puncture are similar, therefore evaluating the levels of TM in serum and CSF dynamically can be used to assess the extracranial and intracranial treatment effect, respectively.@*CONCLUSIONS@#Our study demonstrates that Serum and CSF TM can work as an auxiliary clinical diagnostic tool, which has a potential value in early diagnosis of NSCLC patients with LM. Serial measurement of TM may play an important role in the clinical management of NSCLC patients with LM, which is worthy of further promotion and clinical application.
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BACKGROUND@#Leptomeningeal metastasis (LM) are a severe complication of non-small cell lung cancer (NSCLC), and normally accompanied by poor prognosis. For the patients with targetable mutations, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are the preferred treatment, but the acquired TKI resistance is inextricable. The aim of this study is to analyze the different gene mutation spectrum and mutation frequency of the cerebrospinal fluid (CSF) and plasma in NSCLC patients with LM, and screen out the drug-resistant mutations so as to guide the choice of treatment accurately.@*METHODS@#The paired CSF and plasma samples were collected from the NSCLC-LM patients with acquired TKI resistance. Next generation sequencing (NGS) was used to detect the gene variations of circulating tumor DNA (ctDNA).@*RESULTS@#A total of 18 NSCLC patients with LM were collected. Of the basic mutations, 11 cases (61.11%) were EGFR, 6 cases (33.33%) were anaplastic lymphoma kinase (ALK), and 1 case (5.56%) was ROS proto-oncogene 1, receptor tyrosine kinase (ROS1). Tumor protein p53 gene (TP53) and mesenchymal-epithelial transition factor (MET) were the two most frequently accompanying mutated genes in CSF ctDNA. The detected mutation rate of CSF samples was higher than that of plasma samples (100.00% vs 66.67%, P=0.006), and the maximum allelic fractions were all higher in CSF than in plasma (P<0.001). Abundant single-nucleotide variations (SNV) and copy number variants (CNV) were detected in CSF, the amount of both of which were more than in blood. In addition, the CSF and plasma samples of patients treated with several TKIs had more SNV mutations than patients who received only a single TKI treatment.@*CONCLUSIONS@#For the patients of NSCLC, ctDNA in CSF could reveal genomic alterations of LM more exactly and overally than it in plasma, thus could be an optimal source of liquid biopsy for guiding therapy, monitoring therapeutic effect, and predicting prognosis.
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BACKGROUND: Conventional anterior debridement and bone graft fusion for lumbar spinal tuberculosis have a great trauma, and bring more complications. The double titanium mesh support bone graft combined with posterior pedicle internal fixation reconstruction can significantly improve the prognosis of lumbar spinal tuberculosis. There is no clinical study to compare the efficacy between the two surgical methods. OBJECTIVE: To compare the efficacy of lumbar spinal tuberculosis via anterior double titanium mesh support bone graft combined with posterior pedicle internal fixation and conventional anterior debridement and bone graft fusion. METHODS: Case history data of 40 patients with lumbar spinal tuberculosis were retrospectively collected from the Department of Spinal Surgery, Mianyang Central Hospital, Southwest Medical University from May 2015 to March 2018. The patients were divided into experimental group and control group (n=20) according to the operation. Patients in the experimental group were treated with the anterior double titanium mesh support bone graft combined with the posterior pedicle screw fixation reconstruction. Patients in the control group were treated with anterior debridement and bone graft fusion. All patients signed the informed consent. This study was approved by the Hospital Ethics Committee. RESULTS AND CONCLUTION: (1) Lumbar spinal tuberculosis could be effectively treated with both surgical methods. (2) Compared with the control group, the operation time was shorter; the intraoperative blood loss was less; and the bone graft fusion was faster in the experimental group. (3) With prolongation of the postoperative time, the erythrocyte sedimentation rate and the sagittal Cobb angle of the lesion segment gradually decreased in the two groups. The erythrocyte sedimentation rate and the sagittal Cobb angle of the lesion segment in the experimental group were slightly lower than those in the control group. (4) After treatment, the classification of the American Spinal Cord Injury Association was improved in some patients. (5) The incidence of adverse reactions in the experimental group was lower than that of the control group. (6) The results suggest that double titanium mesh support bone graft combined with posterior pedicle internal fixation reconstruction can effectively improve the stability of the diseased vertebrae, and the treatment effect on lumbar spinal tuberculosis is better than conventional anterior lesion removal and bone graft fusion internal fixation.
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To evaluate the white-matter integrity and its correlation with cognitive function in patients with mild cognitive impairment (MCI) complicated with lacunar infarctions (LI) by diffusion tensor imaging (DTI) of magnetic resonance (MR). Methods: Twenty-six patients with MCI were selected including 14 patients with combined LI and 12 patients without combined LI, and 16 healthy volunteers were recruited as normal control. All subjects completed cognitive function assessment and DTI sequence of MR. Factional anisotropy (FA) and mean diffusivity (MD) values among the MCI with LI group (MCI-LI), the MCI without LI group (MCI-non LI), and the normal control group (NC) were compared, and the correlation between DTI parameters and cognition was determined by multiple linear stepwise regression. Results: Compared with the MCI-non LI group, the FA values were significantly decreased (P<0.05) and MD values were significantly increased (P<0.05) in the white matter fiber bundles (such as the left hippocampus of the cingulate tract, the frontal fascicle of the corpus callosum, the right inferior frontal occipital fascicle, and the right superior longitudinal fascicle) in the MCI-LI group. In the MCI-LI group, the FA value of right cingulate gyrus was significantly correlated with Activity of Daily Living Scale (ADL) score (B=-50.2, 95% CI -77.7 to -22.7, P=0.003); the FA value of left anterior thalamic radiation (B=443.8, 95% CI 222.9 to 664.8, P=0.001) and MD value of left inferior longitudinal tract (B=-318.5, 95% CI -534.7 to -102.3, P=0.009) were significantly correlated with Wechsler digit symbol substitution (WDSS) score; the FA value of left superior temporal lobe longitudinal tract was significantly correlated with Backward Digit Span (BDSP) score (B=12.5, 95% CI 1.5 to 23.4, P=0.030). Conclusion: The integrity of white matter is significantly destroyed in MCI patients with LI than that in MCI patients without LI, and there is a correlation between cognitive function and DTI parameters in some white matter tracts in MCI patients with LI.
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Humans , Anisotropy , Brain , Cognitive Dysfunction , Diagnostic Imaging , Diffusion Magnetic Resonance Imaging , Diffusion Tensor Imaging , Neuropsychological Tests , Stroke, Lacunar , White MatterABSTRACT
Leptomeningeal metastasis (LM) is one of the most severe complications of non-small-cell lung cancer (NSCLC), and its incidence is increasing gradually with the progress of targeted therapies. There are currently no standard guidelines for the therapy of LM. Intrathecal chemotherapy is the mainstay of treatment for NSCLC patients with LM, but the optimal drug, administration route and mode, and dosage remain unclear. We report a case of LM from NSCLC, who received the intrathecal chemotherapy with pemetrexed by Ommaya reservoir after prior targeted therapies. This local treatment improved the quality of life, and obtained the clearing of CSF cytology and stable lesions of LM without any notable side effects. After confirmation of LM, the patient has survived 17 months until now. Here we report the first case to demonstrate the potential effectiveness of intrathecal pemetrexed by Ommaya reservoir for the treatment of LM of NSCLC, summarize the safety and effectiveness of intrathecal chemotherapy in combination with related literatures, and provide a new strategy for local treatment of LM in clinical. .
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Objective To evaluate the efficiency of rotational atherectomy (RA) with drug-eluting stents (DES) by intravascular ultrasound (IVUS) for elderly patients with heavily calcified lesions. Methods Thirteen patients aged above 70 years with heavily calcified coronary artery having underwent the treatment of RA and DES by IVUS were enrolled. The levels of troponin T, creatine kinase isozyme-MB and creatinine were detected before and after operation.The levels of minimus lumen diameter, diameter stenosis rate, effective area of the lumen before and after operation were compared. The immediate success rate was evaluated and the follow-up results were recorded. Results Thirteen patients in accordance with angiography and IVUS examination were confirmed as severe calcification. The levels of troponin T, creatine kinase isozyme-MB and creatinine detected before and after operation had no significantly differences (P>0.05). The levels of minimus lumen diameter, diameter stenosis rate and effective area of the lumen before and after operation had significant differences:(2.06 ± 0.38) mm vs. (3.98 ± 0.76) mm, (73.26 ± 7.02)%vs. (17.00 ± 3.34)%, (4.53 ± 1.50) mm2 vs. (12.54 ± 6.19) mm2, P<0.01. The rate of left anterior descending artery, circumflex artery and right coronary artery calcification was 10/13, 0 and 1/13. All patients′operation was successful. Followed up for (14.6 ± 3.4) months, no patients had angina and myocardial infarction, or required target vessel revascularization. Conclusions RA with DES by IVUS can be safely used in elderly patients with severely calcified coronary artery disease, and can improve the success rate of intervention operation.
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Objective To investigate the metabolic changes and the metabolites distribution of different hippocampal regions (head,body and tail)in patients with Alzheimer’s disease (AD)using 1 H-MRS for early diagnosis.Methods The hippocampal multivoxel 1H-MRS at 3.0 T was scanned in 30 patients with AD and other 30 normal cognitive elders (NC)as contrast.The data obtained were processed at a workstation.The hippocampus was divided into 3 parts (head,body and tail),and the ratios of N-acetylaspartate (NAA)/creatine (Cr),myoinositol (MI)/Cr and MI/NAA were calculated respectively.The metabolite ratios and distribution changes were compared between group AD and NC.Results Compared with the group NC,the NAA/Cr in group AD in bilateral hippocampal body and tail was decreased,whereas the MI/Cr and MI/NAA in bilateral body and tail,MI/NAA in left head were opposite (P0.05).Conclusion Metabolic changes and disappearance of the normal distribution in different hippocampal regions detected by 1 H-MRS provide helpful clues for early diagnosis of the AD.
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<p><b>OBJECTIVE</b>To explore the method for the isolation, cultivation, and purification of adipose-derived stem cells (ADSCs) and examine the oncogenesis and homing of ADSCs to the liver in vivo.</p><p><b>METHODS</b>ADSCs were isolated from female mice by digestion with 0.075% collagenase I and the morphology of the isolated cells was observed with examination of the cell surface markers and cell cycle. BALB/c mice were injected with 1×10(6) ADSCs on the back to evaluate the oncogenesis of ADSCs or with 1×10(6) ADSCs stained with 5, 6-carboxyfluorescein diacetate-succinimidyl ester (CFSE) via the tail vein to examine the cell homing to the liver.</p><p><b>RESULTS</b>The isolated ADSCs highly expressed CD29 and CD44 and were negative for CD34, CD45, CD11b and CD14. Cell cycle distribution analysis showed cell percentages in G0/G1, S, and G2/M phases of 80.1%, 7.9%, and 12%, respectively. The ADSCs had a low immunogenicity and did not express CD40, CD80, CD86, MHCI, MHCII or PDL-1. After stimulation with IFN-γ, the expression of CD40, CD80 and PDL-1 were up-regulated slightly in the cells. Dorsal injection of the ADSCs did not result in any tumor formation within 1 month, and ADSCs injected via the tail vein showed cell homing to the liver.</p><p><b>CONCLUSION</b>Murine ADSCs can be isolated and expanded effectively by collagenase digestion and adherent culture. The isolated ADSCs can successfully reside in the liver after implantation, and thus may serve as a promising candidate cell in stem cell therapy of liver diseases.</p>
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Animals , Female , Mice , Adipocytes , Cell Biology , Cell Culture Techniques , Cell Differentiation , Cell Movement , Cells, Cultured , Liver , Cell Biology , Mesenchymal Stem Cells , Cell Biology , Mice, Inbred BALB C , Stem Cell TransplantationABSTRACT
Objective Accumulating reports have suggested that hepatic stellate cells (HSCs) exhibit immunosuppressive ability and may be responsible for the occurrence and development of hepatocellular carcinoma (HCC).The mechanisms through which HSCs affect T-cell-induced adaptive immune responses and the relationship with the regulatory T cells (Treg cells) were studied.Methods We isolated HSCs from wildtype mice to demonstrate the influence of HSCs on T-cell proliferation and explored their effect on Treg cells through mixed leukocyte reactions (MLRs) in vitro.Results We found that activated HSCs could induce T-cell hyporesponsiveness in adaptive immune response by inhibiting the proliferation of T cells andincreasing the quantity of Treg cells.Conclusion Activated HSCs may lead to hypoergia of T cells in adaptive immune reaction and up-regulate the expression of Treg cells,thus facilitating immunotolarance.
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To investigate the influence of bear bile on rat hepatocarcinoma induced by diethylnitrosamine (DEN), a total of 40 rats were randomly divided into 4 groups: normal control group, model group, and two bear bile treatment groups. The rat liver cancer model was induced by breeding with water containing 100 mg x L(-1) DEN for 14 weeks. The rats of the bear bile groups received bear bile powder (200 or 400 mg x kg(-1)) orally 5 times per week for 18 weeks. The general condition and the body weight of rats were examined every day. After 18 weeks the activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and total bilirubin (TBIL) were detected. Meanwhile, the pathological changes of liver tissues were observed after H&E staining. The expression of proliferative cell nuclear antigen (PCNA) and a-smooth muscle actin (alpha-SMA) in liver tissue were detected by immunohistochemical method. After 4 weeks the body weights of rats in normal group were significantly more than that in other groups (P < 0.05); and that in the two bile groups was significantly more than that in the model group. Compared with normal group, the level of serum glutamic-pyruvic transaminase and total bilirubin increased significantly in other groups; compared with model group, these two indexes decreased significantly in two bile groups. Hepatocellular carcinoma occurred in all rats except for normal group; there were classic cirrhosis and cancer in model group while there were mild cirrhosis and high differentiation in two bile groups. There were almost no expressions of PCNA and alpha-SMA in normal group while there were high expressions in model group; the two bile groups had some expressions but were inferior to the model group, and alpha-SMA reduced markedly. It indicated that bear bile restrained the development of liver cancer during DEN inducing rat hepatocarcinoma, which may be related to its depressing hepatic stellate cell activation and relieving hepatic lesion and cirrhosis.
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ObjectiveTo isolate and culture human adipose derived mesenchynal stem cells (hADSCs) and study the potential of hADSCs to differentiate into hepatocyte-like cells. MethodsTo ensure the removal of contaminating hematopoietic cells, hADSCs were selected based on plastic adherence. Cell surface antigen was confirmed by flow cytometry; ultramicrostructure was detected by transmission electron microscopy; adipogenic and osteogenic differentiation of hADSCs was analyzed by oil Red O staining and yon Kossa staining. hADSCs were exposed to differentiation medium containing EGF,FGF,OSM, HGF,TSA in vitro. 10% CCl4 (100 μ1/20 g body weight )was injected into immune-deficient BALB/c-nu mice and hADSCs (5 × 105 cells) were simultaneously administrated from the tail vein. Blood samples were collected and concentration of aminotransferase and direct bilirubin were detected. Administration without hADSCs was used as a control. One month later, we sacrificed the mice and liver sections were examined by histochemical immunofluorescence with human ALB specific antibodies. ResultshADSCs exhibited fibroblast-like morphology, and expressed CD73, CD90,CD105, and were lacking of CD34 and CD45. Adipogenic and osteogenic differentiation showed that hADSCs have the capacity of multidifferentiation. The differentiated cells showed hepatocyte-like cell morphologies and hepatocyte-specific markers including albumin (alb) and α-fetoprotein (AFP). The bioactivity assays revealed that these hepatocyte-like cells could uptake low-density lipoprotein (LDL).Histochemical immunofluorescence showed that hADSCs were incorporated into injured livers. Some human alb-positive cells were found in liver sections after implantation of undifferentiated hADSCs. Transaminase activity in the experimental group was lower than in the control group. Conclution hADSCs can differentiated into functional hepatocyte-like cells and can relieve CCl4 induced BALB/c-nu acute liver injury.
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Objective To determine immune modulatory activity of activated hepatic stellate cells( HSCs) in hepatocellular carcinoma and immune response in tumor microenvironment. Methods Cell proliferation was measured by BrdU incorporation with a microtiter plate reader at 450 nm. The effect of HSCs on T cell proliferation was measured by MLR. Mouse hepatic cancer cell line H22 were implanted on the backs of BALB/c mice to establish the subcutaneous transplanted tumor model. Then the mice were sacrificed after 20 days for anatomical and size determination. Furthermore, Paraffin-embedded tissue was removed by serial tissue sectioning and immunohistochemically examined for expression of T lymphocyte subsets. T lymphocyte subsets in splenocytes were detected by FCM. Apoptotic mononuclear cells were evaluated by FITC-labeled Tunel assay. Results We determined that HSCsCM promoted hepatocellular carcinoma(HCC) cell line proliferation and HSCs inhibit T cell proliferation by MLR in vitro. We also examined normal immune mice to assess the immunosuppression of HSCs in the development of HCC. In the co-transplantation with HSCs group, T cells and their subtypes decreased obviously in the tumors and the spleen. The results showed that co-transplanted HSCs can induce more PD-L1 expression and more mononuclear cell apoptosis in tumor tissue. Conclusion Our results demonstrated that HSCs promote HCC progression partially because of their immune regulatory activity. Our data supply new information to support an integral role for HSCs in promoting HCC progression and suggest that HSCs may serve as a therapeutic target for HCC.
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Objective To investigate the cytotoxic effects and mechanism of PNP-CD chimeric gene vector originated from PNP/MeP-dR system on HCC cells. Methods The fusion suicide gene PNP-CD obtained by site directed mutagenesis technique was subcloned into pcDNA3.0 to construct a eukaryotic expression vector containing a chimeric gene, pcDNA3.0/ PNP-CD. After being identified by recombinant enzyme, PCR and subsequent sequencing, it was transfected into HepG2 cells by liposome-mediation method. The G418-resistant cellular clone with stable transfection of pcDNA3.0/PNP-CD, HepG2/PNP-CD was established by selection. The expression of PNP-CD gene was also verified by RT-PCR and Western blotting. The curve of cellular growth was assayed by Trypan blue exclusion. The cellular sensitivity of HepG2/PNP-CD to its specific prodrugs and its bystander effects were also assayed by MTT method. Results The chimeric gene, PNP-CD, was inserted into pcDNA3.0 correctly, and the stable expression of pcDNA3.0/PNP-CD in HepG2 cells was confirmed.This cellular clone was highly sensitive to its corresponding prodrugs. It was indicated that its bystander effects with the synergetic treatment of its specific prodrugs were substantially higher than those caused by the same vector with the administration of only a single prodrug, MeP-dR. Conclusion The bi-functional fusion suicide gene vector, pcDNA3.0/PNP-CD, yields powerful cytotoxic effects on HCC cells in the presence of the synergetic treatment of its specific prodrugs, which would be a high-performance therapeutic vector in gene therapy for liver cancer.
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Objective To isolate and indentify fetal hepatic progenitor/stem cells, and study the feasibility and effectiveness of their transplantation on acute liver injury in nude mice. Methods The primitive cells isolated from 13.5dpc pregnant mouse fetal hver by way of enzyme digesting were cultured in vitro and liver specific markers as AFP, CK19, Albumin, c-Met, were identified by immunofluorescence and RT-PCR on colonies. 4 × 105 cells were transplanted into nude mice with carbon tetrachloride induced acute liver injury. Hapatic functions were measured pre- and -post transplantation on days 1,2,4, 7. Meanwhile, hepatic pathology was studied. Results Compared to control group the hepatic functions gradually recovered in transplant group, on days 1,2,4 after fetal hepatic stem cell transplantation. The hepatic pathology significantly improved in stem cells transplantation group. Conclution Fetal hepatic progenitor/stem cell were successfully yielded by enzyme digest. Stem cells transplantation improved the hepatic function and pathology in acute hapatic injury model of nude mice.
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Objective To study the effects of liver graft on the immune tolerance of intestinal allograft in auxiliary en-bloc liver-small bowel transplantation in pigs.Methods Seventy outbreed Landrace pigs were divided into 4 groups.Ten auxiliary liver-small bowel allotransplantations were performed in group A,B,C,respectively,and 5 segmental small bowel allotransplantatiom were performed in group D.Pigs were administered with routine and lower dose of cyclosporine and methylprednisolone in group B and C,respectively. No immunosuppressive agent was administered to pigs in group A and D.Results The initial time of acute rejection was obviously prolonged in group A than group D.and the acute rejection was milder in group A than group D(P＜0.05).There was no significant difference upon postoperative survival time,initial time of acute rejection and degree of acute rejection between group B and C(P＞0.05).Conclusions The immune tolerance of intestinal allograft Can be induced by liver graft in auxiliary en-bloc liver-small howel transplantation.
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ObjectiveTo set up the porcine combined liver/small bowel orthotopic allotransplantation model.MethodThe donors including liver, about 2 to 3?m proximal jejunum, duodenum and a sma ll part of pancreas head were transplanted to the hybridized long-white recipie nt pigs after the resection of their livers and the majority of small bowels.ResultWithout using immunosuppressive drugs, the median survival time of the animals w as 92?h with the longest survival time being 196?h, and there was about 75?% of the animals lived for more than 24?h after operation.ConclusionThe successful establishment of the combined liver/small bowel transplantation m odel will do great favor to our future clinical practice and further study of th e combined liver/small bowel transplantation.