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OBJECTIVE@#To assess the value of single sperm sequencing in preimplantation genetic testing for monogenic disease (PGT-M).@*METHODS@#A Chinese couple with two children whom had died of Spinal muscular atrophy (SMA) and attended the Jiangxi Provincial Maternal and Child Health Care Hospital in June 2020 was selected as the subject. Eleven single sperm samples were isolated by mechanical immobilization and subjected to whole genome amplification. Real-time PCR and Sanger sequencing were used to detect the SMN1 variants in the single sperm samples. Genomic DNA of the wife, her parents and the husband, as well as one single sperm sample harboring the SMN1 variant and two single sperm samples without the variant were used for the linkage analysis. Targeted capture and high-throughput sequencing were carried out to test 100 single nucleotide polymorphisms distributed within 2 Mb up- and downstream the variant site. The haplotypes linked with the SMN1 variants were determined by linkage analysis. Blastocyst embryos were harvested after fertilizing by intracytoplasmic sperm injection. Cells from the trophoblasts of each embryo were biopsied and subjected to whole genome amplification and targeted capture and high-throughput sequencing to determine their carrier status. Chromosomal aneuploidy of wild-type embryos was excluded. An euploid embryo of high quality was transferred. Amniotic fluid sample was taken at 18 weeks of gestation to confirm the status of the fetus.@*RESULTS@#Genetic testing showed that the couple both had deletion of exons 7 ~ 8 of the SMN1 gene. The wife has inherited the deletion from her father, while the husband was de novo. The haplotypes of the husband were successfully constructed by single sperm sequencing. Preimplantation genetic testing has indicated that 5 embryos had harbored the heterozygous variant, 4 embryos were of the wild type, among which 3 were euploid. Prenatal diagnosis during the second trimester of pregnancy has confirmed that the fetus did not carry the deletion.@*CONCLUSION@#By single sperm sequencing and PGT-M, the birth of further affected child has been successfully avoided.
Subject(s)
Humans , Pregnancy , Female , Child , Male , Preimplantation Diagnosis , East Asian People , Semen , Genetic Testing , Muscular Atrophy, Spinal/genetics , Aneuploidy , Blastocyst/pathology , High-Throughput Nucleotide Sequencing , SpermatozoaABSTRACT
Clinical and endoscopic data of 6 patients with colorectal mucosa associated lymphoid tissue (MALT) lymphoma who were diagnosed by endoscopy in the Digestive Endoscopy Center of Jiangsu Province Hospital of Chinese Medicine from January 2015 to June 2021 were retrospectively analyzed. There were 2 males and 4 females with aged from 62 to 87 years. The lesions were located in rectum in 3 cases, transverse colon in 1 case, sigmoid colon in 1 case, and sigmoid colon and rectum in 1 case. There were 1 case of polyposis type, 2 cases of inflammation type, and 3 cases of submucosal tumor type. The "tree-like appearance (TLA)" found in 5 cases. Endoscopic resection, surgery combined with chemotherapy, Helicobacter pylori eradication and follow-up were performed on 2, 1, 1 and 2 cases, respectively. Five cases had a good prognosis after 21-73 months follow-up, and 1 case had lost to follow-up. No recurrence was found in endoscopic and imaging review. Colorectal MALT lymphoma should be considered when colonoscopy detects a submucosal lesion with TLA sign on the left colon. Endoscopic resection has the potential to be a first-line treatment in the context of early diagnosis.
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@#AIM: To evaluate the clinical efficacy of local application of triamcinolone acetonide combined with mouse nerve growth factor in the treatment of infraorbital nerve injury after infraorbital wall fracture.METHODS: Forty-three patients(43 eyes)with infraorbital wall fractures who underwent infraorbital wall fracture revision from April 2020 to February 2021 at the Affiliated Eye Hospital of Nanchang University were prospectively analyzed. Patients were randomly divided into two groups, in which 20 patients(20 eyes)in the experimental group had gelatin sponges infiltrated with triamcinolone acetonide and mouse nerve growth factor placed on the nerve injury intraoperatively; 23 patients(23 eyes)in the control group had no special treatment intraoperatively. At 6mo postoperative follow-up, the results of quantitative sensory testing(two-point localization, nociception, and touch)were compared between the affected and healthy lower lid areas, and the results were reported in an asymmetry index(AI).RESULTS: Baseline results showed no significant differences between the two groups in terms of gender, age, time of injury, and preoperative sensory testing between the two groups(all <i>P</i>>0.05). The AI values of two-point localization sensation, tactile sensation, and pain sensation in both groups were higher at 1wk after surgery than before surgery(all <i>P</i><0.05), and the symptoms of sensory impairment were aggravated, with different degrees of improvement at 1mo after surgery and statistically significant differences in pain sensation at 3mo after surgery(<i>P</i><0.05), and two-point localization sensation, tactile sensation, and pain sensation were significantly improved at 6mo after surgery than before treatment(all <i>P</i><0.01). At 1mo after surgery, the differences in two-point localization sensation and pain sensation in the test group were statistically significant compared with the control group(<i>t</i>=-2.082,-2.143; <i>P</i>=0.044, 0.038). At 3mo after surgery, there was a statistically significant difference in nociception in the test group compared to the control group(<i>t</i>=-2.118, <i>P</i>=0.04). At 6mo after surgery, there was no statistically significant difference in quantitative sensory testing between the two groups(<i>P </i>>0.05).CONCLUSION: Local application of triamcinolone acetonide combined with mouse nerve growth factor for the treatment of infraorbital nerve injury after infraorbital wall fracture was effective in early internal recovery and superior to the group without special intraoperative treatment.
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Objective: To explore the combination rule of Corydalis Rhizoma-Toosendan Fructus drug pair through the research in "Pharmaceutical Standard of the Ministry of Health of the People's Republic of China-Traditional Chinese Medicine Formulation", so as to provide theoretical basis for clinical application and new product development of this pair. Methods: The prescriptions containing Corydalis Rhizoma-Toosendan Fructus were collected from Pharmaceutical Standard of Ministry of Public Health of the People's Republic of China-TCM Formulation and input into TCM inheritance support platform software (V2.5) to establish the database. The frequency of attending diseases, compatibility medicinal materials, and meridian of drug were analyzed statistically. The core combination of medicinal materials in the prescriptions containing Corydalis Rhizoma-Toosendan Fructus were analyzed statistically with association rule Apriori algorithm (support degrees were 10%, 20%, 30%, confidence was 0.90). Meridian of drug with higher compatibility frequency were selected and analyzed in respect of prescription rules. Results: A total of 51 prescriptions containing Corydalis Rhizoma-Toosendan Fructus were screened, eight kinds of attending diseases (frequency≥2), such as stomachache, hypochondriac pain, leukorrhagia, dysmenorrhea, pelvic inflammation, menstrual disorder, etc. Nine kinds of attending syndrome (frequency≥2) were included such as liver-stomach disharmony, syndrome of qi stagnation and blood stasis, syndrome of liver depression and qi stagnation, syndrome of liver depression and blood deficiency, syndrome of blood-heat in uterus, etc. There were 17 commonly used compatibility medicinal materials (frequency≥5), including Paeoniae Radix Alba, Aucklandiae Radix, Glycyrrhizae Radix et Rhizoma, Cyperi Rhizoma etc. There were 20 commonly used medicinal materials combinations (frequency≥6), including Corydalis Rhizoma-Paeoniae Radix Alba-Toosendan Fructus, Corydalis Rhizoma-Aucklandiae Radix-Toosendan Fructus, Corydalis Rhizoma-Glycyrrhizae Radix et Rhizoma-Toosendan Fructus, Corydalis Rhizoma-Cyperi Rhizoma-Toosendan Fructus. Meridian of drug with higher compatibility frequency was distributed to liver, spleen and stomach channel tropism. There were five kinds of core medicinal materials commonly acted on the liver meridian with Corydalis Rhizoma-Toosendan Fructus, including Aucklandiae Radix, Paeoniae Radix Alba, Citri Reticulatae Pericarpium, Amomi Fructus, and Aquilariae Lignum Resinatum. There were commonly used core medicinal materials combinations (frequency≥7), including Corydalis Rhizoma-Aucklandiae Radix-Toosendan Fructus, Corydalis Rhizoma-Paeoniae Radix Alba-Toosendan Fructus, Corydalis Rhizoma-Citri Reticulatae Pericarpium-Toosendan Fructus, Corydalis Rhizoma-Citri Reticulatae Pericarpium-Paeoniae Radix Alba-Aucklandiae Radix-Toosendan Fructus. There were three kinds of core medicinal materials commonly acted on the spleen meridian and the stomach meridian with Corydalis Rhizoma-Toosendan Fructus, including Aquilariae Lignum Resinatum, Aucklandiae Radix and Amomi Fructus. There were commonly used core medicinal materials combinations (frequency≥4), including Corydalis Rhizoma-Aquilariae Lignum Resinatum-Toosendan Fructus, Corydalis Rhizoma-Aucklandiae Radix-Toosendan Fructus, Corydalis Rhizoma-Amomi Fructus-Toosendan Fructus. Conclusions: Herbal medicines used frequently in prescriptions containing Corydalis Rhizoma-Toosendan Fructus can regulate qi flowing for relieving pain. The medicine using is relatively concentrated, and the composition is clear. In addition, Prescription rules of three kinds of meridian prescriptions were chosen to make comparative analysis, and the compatibility features of the prescriptions containing Corydalis Rhizoma-Toosendan Fructus was reflected in different meridian of drug and the composing principles. This study also can provide theoretical basis for treating different diseases with same method and the better application of Corydalis Rhizoma-Toosendan Fructus in clinical practice.
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OBJECTIVE@#To delineate the variants spectrum of phenytalanine hydroxylase (PAH) gene among 78 unrelated patients with phenylketonuria (PKU) from Jiangxi province.@*METHODS@#The 13 exons and flanking intronic regions of the PAH gene were subjected to PCR amplification and sequencing.@*RESULTS@#A total of 143 variants were detected among the 156 alleles, which included 54 types of variants, which yielded a detection rate of 91.7%. Common variants have included R243Q (26/143, 18.2%), R408Q (10/143, 7.0%), EX6-96A to G(8/143, 5.6%), IVS4-1G to A(7/143, 4.9%), R241C(7/143, 4.9%) and V399V(7/143, 4.9%). In addition, 6 novel variants were detected, which included IVS4-3T to G, Q172H, C284Y, V291L, V329del, and L430R. The variants consisted of missense, splicing, nonsense and deletion variants, which have mainly located in exons 7 (45, 31.5%), 12(17, 11.9%), 11(16, 11.2%) and 6(14, 9.8%).@*CONCLUSION@#Variants of the PAH gene identified in Jiangxi province mainly involve exons 7, 12, 11 and 6, with the most common variants being R243Q and R408Q. Six novel variants were identified.
Subject(s)
Humans , China , Exons , Introns , Mutation , Phenylalanine Hydroxylase , Genetics , Phenylketonurias , GeneticsABSTRACT
Objective@#To delineate the variants spectrum of phenytalanine hydroxylase (PAH) gene among 78 unrelated patients with phenylketonuria (PKU) from Jiangxi province.@*Methods@#The 13 exons and flanking intronic regions of the PAH gene were subjected to PCR amplification and sequencing.@*Results@#A total of 143 variants were detected among the 156 alleles, which included 54 types of variants, which yielded a detection rate of 91.7%. Common variants have included R243Q (26/143, 18.2%), R408Q (10/143, 7.0%), EX6-96A>G (8/143, 5.6%), IVS4-1G>A (7/143, 4.9%), R241C (7/143, 4.9%) and V399V (7/143, 4.9%). In addition, 6 novel variants were detected, which included IVS4-3T>G, Q172H, C284Y, V291L, V329del, and L430R. The variants consisted of missense, splicing, nonsense and deletion variants, which have mainly located in exons 7 (45, 31.5%), 12 (17, 11.9%), 11 (16, 11.2%) and 6 (14, 9.8%).@*Conclusion@#Variants of the PAH gene identified in Jiangxi province mainly involve exons 7, 12, 11 and 6, with the most common variants being R243Q and R408Q. Six novel variants were identified.
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Background@#Chronic obstructive pulmonary disease (COPD) and obstructive sleep apnea (OSA) syndrome are highly prevalent respiratory conditions. Their coexistence is referred to as the overlap syndrome. They are both related to pulmonary hypertension (PH) development. This study investigated the effects of OSA on PH in patients with COPD and the associated factors.@*Methods@#Consecutive patients with stable COPD were recruited for an observational cross-sectional study from September 2016 to May 2018 at Peking University Third Hospital. In total, 106 patients with COPD were enrolled and performed home portable monitoring and echocardiography. OSA was defined by an apnea hypopnea index (AHI) ≥10 events/h. Based on OSA absence or presence, patients were divided into the COPD with OSA and COPD without OSA groups. Factors affecting pulmonary artery pressure (PAP) and PH were identified using univariate analysis and logistic regression models.@*Results@#In the 106 patients with COPD, the mean age was 69.52 years, 91.5% were men, and the mean forced expiratory volume in 1 s (FEV1) percentage of predicted was 56.15%. Fifty-six (52.8%) patients with COPD were diagnosed with OSA, and 24 (22.6%) patients with COPD were diagnosed as PH. Compared with COPD without OSA group, the median PAP in COPD with severe OSA group increased by 5 mmHg (36.00 [26.00–50.00] mmHg vs. 31.00 [24.00–34.00] mmHg, P = 0.036). COPD with percent of night-time spent with oxygen saturation below 90% (T90) > 10% group had higher PAP than COPD with T90 ≤ 1% group (36.00 [29.00–50.00)] mmHg vs. 29.00 [25.50–34.00] mmHg, F = 7.889, P = 0.007). Univariate analysis revealed age, FEV1% predicted, T90, and Charlson index had statistically significant effects on PH. Multiple regression analysis showed a significant and independent effect of both FEV1% predicted (odds ratio [OR] = 3.46; 95% confidence interval [CI]: 1.15–10.46; P = 0.028) and AHI (OR = 3.20; 95% CI: 1.09–19.35; P = 0.034) on PH.@*Conclusions@#Patients with COPD with OSA are more susceptible to PH, which is associated with declining lung function and increased severity of OSA. Thus, nocturnal hypoxemia and OSA in elderly patients with COPD should be identified and treated.
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BACKGROUND@#Chronic obstructive pulmonary disease (COPD) and obstructive sleep apnea (OSA) syndrome are highly prevalent respiratory conditions. Their coexistence is referred to as the overlap syndrome. They are both related to pulmonary hypertension (PH) development. This study investigated the effects of OSA on PH in patients with COPD and the associated factors.@*METHODS@#Consecutive patients with stable COPD were recruited for an observational cross-sectional study from September 2016 to May 2018 at Peking University Third Hospital. In total, 106 patients with COPD were enrolled and performed home portable monitoring and echocardiography. OSA was defined by an apnea hypopnea index (AHI) ≥10 events/h. Based on OSA absence or presence, patients were divided into the COPD with OSA and COPD without OSA groups. Factors affecting pulmonary artery pressure (PAP) and PH were identified using univariate analysis and logistic regression models.@*RESULTS@#In the 106 patients with COPD, the mean age was 69.52 years, 91.5% were men, and the mean forced expiratory volume in 1 s (FEV1) percentage of predicted was 56.15%. Fifty-six (52.8%) patients with COPD were diagnosed with OSA, and 24 (22.6%) patients with COPD were diagnosed as PH. Compared with COPD without OSA group, the median PAP in COPD with severe OSA group increased by 5 mmHg (36.00 [26.00-50.00] mmHg vs. 31.00 [24.00-34.00] mmHg, P = 0.036). COPD with percent of night-time spent with oxygen saturation below 90% (T90) > 10% group had higher PAP than COPD with T90 ≤ 1% group (36.00 [29.00-50.00)] mmHg vs. 29.00 [25.50-34.00] mmHg, F = 7.889, P = 0.007). Univariate analysis revealed age, FEV1% predicted, T90, and Charlson index had statistically significant effects on PH. Multiple regression analysis showed a significant and independent effect of both FEV1% predicted (odds ratio [OR] = 3.46; 95% confidence interval [CI]: 1.15-10.46; P = 0.028) and AHI (OR = 3.20; 95% CI: 1.09-19.35; P = 0.034) on PH.@*CONCLUSIONS@#Patients with COPD with OSA are more susceptible to PH, which is associated with declining lung function and increased severity of OSA. Thus, nocturnal hypoxemia and OSA in elderly patients with COPD should be identified and treated.
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Exosomes are vesicle structures containing a variety of proteins, mRNA and Micro RNA and other important signaling molecules, which can participate in the exchange of materials and information between cells, thus affecting the physiological functions of cells. Tumor growth and metastasis depend on adequate angiogenesis and blood supply. More and more studies have confirmed that tumor drived exosomes are involved in promoting tumor angiogenesis. This article reviews how exosomes drive angiogenesis to add new options for improving existing antiangiogenic therapies.
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Objective To analyze the influencing factors of strain ratio in diagnosing thyroid lesions of thyroid imaging reporting and data system (TI-RADS) 4-6 categories, and explore the application value of elastic strain ratio in differentiating malignant thyroid nodules from benign ones under different influencing factors.Methods A retrospective analysis was made on the pathologic data of 278 nodules undergone TI-RADS ultrasonography stratification and ultrasound elestography, and 105 nodules of TI-RADS 4-6 categories were selected as research objects. A receiver-operating characteristic (ROC) curve was used to identify the cut-off point for differential diagnosis of thyroid nodules. A logistic regression model was established to analyze the factors affecting strain ratio in the differential diagnosis on thyroid nodules of TI-RADS 4-6 categories. Analyses were made in groups to judging the sensitive, specificity, accuracy, positive predictive value and negative predictive of strain ratio under the impact of different factors. Results The best cut-off point of ROC curve for benign and malignant nodules was 0.54. It was found through the logistic regression model that the strain ratio value was closely related to the size, calcification, and thyroid diffuse disease, and strain ration was of higher value in the thyroid nodules which were less than 1 cm without calcification and thyroid diffuse disease.Conclusions Three factors including size, calcification, and diffuse lesions of the thyroid nodules can influence the elastic strain ratio of TI-RADS 4-6 thyroid nodules. Comprehensive analysis of these three influencing factors can improve the application value of elastic strain ratio in differential diagnosis of benign and malignant thyroid nodules.
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Objective To describe the clinical characteristics of a patient with Gitelman syndrome,and to identify the associated SLC12A3 gene mutations.Methods A suspected case of teenager-onset Gitelman syndrome was observed in our hospital.It was further confirmed by clinical manifestations and auxiliary examination.In addition,direct sequencing for the exons of SLC12A3 gene and CLCNKB gene region was conducted to identify the probable disease-associated mutations.Results The case showed characteristics of hypokalemia,hypomagnesemia,and low level of urinary calcium and onset by age of 18.By excluding the possibilities of long-term use of thiazide diuretics,laxatives,chronic vomiting and diarrhea,he was finally diagnosed as a case of Gitelman syndrome.Furthermore,by Sanger direct sequencing,2 coding variations were identified in SLC12A3 gene region,including T304M and L488P.L488P was a new heterozygous mutation.Conclusion Detection of SLC12A3 gene mutation could facilitate the diagnosis of Gitelman syndrome and improve prognosis.
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Salivary analysis can be used to assess the severity of caries. Of the known salivary proteins, a paucity of information exists concerning the role of proteinase 3 (PR3), a serine protease of the chymotrypsin family, in dental caries. Whole, unstimulated saliva was collected from children with varying degrees of active caries and tested using a Human Protease Array Kit and an enzyme-linked immunosorbent assay. A significantly decreased concentration of salivary PR3 was noted with increasing severity of dental caries (P<0.01); a positive correlation (r=0.87; P<0.01; Pearson's correlation analysis) was also observed between salivary pH and PR3 concentration. In an antibacterial test, a PR3 concentration of 250 ng·mL⁻¹ or higher significantly inhibited Streptococcus mutans UA159 growth after 12 h of incubation (P<0.05). These studies indicate that PR3 is a salivary factor associated with the severity of dental caries, as suggested by the negative relationship between salivary PR3 concentration and the severity of caries as well as the susceptibility of S. mutans to PR3.
Subject(s)
Child , Female , Humans , Male , Dental Caries , Myeloblastin , Metabolism , SalivaABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of Shadu Cao Mixture (SDCM, traditional Chinese medicine) on immune functions of immunosuppression mice.</p><p><b>METHODS</b>Fifty BALB/C mice were randomly divided into blank control group, model group, SDCM low-dose, middle-dose and high-dose group. Except the blank control group, other groups were intraperitoneal injected with cyclophosphamide (40 mg/kg) to establish immunosuppression mice model. The blank control group and model group received gavage administration with nonnal saline, while the other groups received gavage administration with different doses of SDCM (10, 20, 40 m/kg for 15 days) respectively. The number of leukocytes and serum levels of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in peripheral blood, spleen index, and the function of NK cells were measured.</p><p><b>RESULTS</b>Compared with the model group , SDCM increased the number of leukocytes and serum concentrations of IL-2, TNF-α and IFN-γ in peripheral blood and improved the spleen index and the function of NK cells significantly (P < 0.05-0.01).</p><p><b>CONCLUSION</b>SDCM could remarkably enhance the immune functions of immunosuppression mice induced by cyclophosphamide.</p>
Subject(s)
Animals , Mice , Cyclophosphamide , Disease Models, Animal , Drugs, Chinese Herbal , Pharmacology , Immunosuppression Therapy , Interferon-gamma , Blood , Interleukin-2 , Blood , Killer Cells, Natural , Allergy and Immunology , Mice, Inbred BALB C , Spleen , Allergy and Immunology , Tumor Necrosis Factor-alpha , BloodABSTRACT
Objective To explore the application value of ultrasound-guided fine needle aspiration biopsy(FNAB) in diagnosing thyroid nodules of TI-RADS 4-6 categories.Methods A retrospective analysis was made on the pathologic data of 494 patients (totally 501 nodules) who underwent TI-RADS ultrasonographic stratification and ultrasound-guided FNAB.168 thyroid nodules of TI-RADS 4-6 categories were selected from 163 patients and classified into three groups:group T,TI-RADS 4A categories;group Ⅱ,TI-RADS 4B category; group Ⅲ,TI-RADS 5-6 category(the pathologically confirmed cases in category 6 had been removed).The ultimate pathologic findings were taken as golden standards to calculate the accuracy,sensitivity,specificity,positive predictive value of ultrasound-guided FNAB in the three groups.ROC curve was drawn to evaluate the diagnostic value of FNAB.Results The accuracy,sensitivity,specificity,positive predictive value of the ultrasound-guided FNAB in the three groups were listed as follows:group Ⅰ 74.4％,73.3％,75.0％,64.7％;group Ⅱ 83.9％,87.5％,71.4％,91.3％;group Ⅲ 89.3％,91.7 ％,75.0％,95.7 ％ respectively.The positive predictive values of the ultrasound-guided FNAB for group Ⅰ and group Ⅱ was higher than that of conventional TI-RADS classification criteria,so the difference was statistically significant; while the positive predictive values for group Ⅲ was close to the conventional ultrasound TI-RADS classification criteria,the difference was not statistically significant.Conclusions The combination application of TI-RADS classification criteria and FNAB can improve the accuracy of ultrasound diagnosis in group Ⅰ and group Ⅱ,and reduce the puncture rate of group Ⅲ.
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Objective To investigate the diagnostic value and clinical significance of the three-dimensional contrast-enhanced MR angiography (3D-CE-MRA)in the infantile superficial hemangioma.Methods Forty-four children with superficial hemangioma un-derwent conventional and dynamic contrast-enhanced MRI.MRI scanning was started at the time of inj ection,and four dynamic pha-ses of images were acquired continually.Maximum intensity proj ection (MIP)images were reconstructed to show the blood vessels of the lesions in multiple phases.Results Forty-nine infantile superficial hemangiomas were detected by MRI,including a single le-sion in 41 patients and multiple ones in 3.3D-CE-MRA showed 37 lesions in 32 patients,and other 12 lesions were not found in 12 patients.Conclusion The conventional and dynamic contrast-enhanced MRI can reflect the location,number and the range of the su-perficial hemangioma,and show the relationship between the lesion and the surrounding tissues.3D-CE-MRA directly shows the blood supply of the tumors.
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Anti-angiogenesis mechanism plays a vital role in tumor targeting immunotherapy. Based on the amino acid sequence of an anti-VEGFR-2 scFv-Fc fusion antibody (AK404R-Fc), this article is aimed to generate an anti-VEGFR-2 human IgG1-like full length antibody (Mab-04). Firstly, the light chain (L-chain) and heavy chain (H-chain) were obtained by overlap PCR and then linked to eukaryotic expression vector pcDNA3.1, separately. The recombinant plasmids (pcDNA3.1-L-chain and pcDNA3.1-H-chain) were then co-transfected into CHO-k cells using liposome transient transfection. Subsequently, Mab-04 antibody was expressed and purified by Protein A affinity chromatography. Western blotting was applied to identify the expression of Mab-04 and its affinity was detected by ELISA assay. DNA sequencing revealed the successful construction of recombinant plasmids and Western blotting assay proved the successful expression of full-length antibody (1 microg x mL(-1)). Finally, ELISA assay illustrated that the binding of the antibody to its antigen was in a concentration-dependent manner (IC50: 50 nmol x L(-1)). These outcomes above indicated that Mab-04 was successfully expressed and assembled, which laid the foundation for further preparation and antineoplastic activity study.
Subject(s)
Animals , Antibodies, Monoclonal, Humanized , CHO Cells , Cricetulus , Genetic Vectors , Immunoglobulin Fc Fragments , Genetics , Immunoglobulin G , Genetics , Allergy and Immunology , Plasmids , Recombinant Fusion Proteins , Genetics , Single-Chain Antibodies , Genetics , Transfection , Vascular Endothelial Growth Factor Receptor-2 , Genetics , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVES</b>To obtain high therapeutic effect and low toxicity single-chain immunotoxin against hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Human mutant tumor necrosis factor-alpha (mTNFalpha) was linked with the 3' end of humanized single-chain Fv against HCC (hscFv25) in pGEX4T-1 vector. The anti-HCC immunotoxin was expressed in Escherichia coli and identified by western blot. The primary tumor regression trial in nude mice bearing HCC was evaluated the targeting therapeutic value of hscFv25-mTNFalpha. The tumor tissues were stained by immunohistochemical with TNFalpha antibody.</p><p><b>RESULTS</b>The expression of single-chain immunotoxin hscFv25-mTNFalpha was 12% of total bacteria proteins. The tumor regression trials of hscFv25-mTNFalpha showed 5/5 effective. It had 2/5 completely remission and 3/5 partly remission. The therapeutic result of hscFv25-mTNFalpha was better than that of mTNFalpha (F=8.70, 0.05). The HCC tissue treated by hscFv25-mTNFalpha expressed TNFalpha positive reaction. The positive granule mainly existed in HCC cytoplasm.</p><p><b>CONCLUSION</b>The single-chain immunotoxin hscFv25-mTNFalpha has high therapeutic effect and low toxicity. It has potentialities for clinical application.</p>