Objective To amplificate,clone and sequence the thymidine kinase (TK) gene of herpes simplex virusⅡ(HSVⅡ); to construct and appraise the fusion gene eukaryon expression vector, pcDNA3/HSVⅡ TK/angiostatin. MethodsThe Hep2 cells were infected by HSVⅡ Sav strain. HSVⅡ genomic DNA was purified from the Hep2 cells suspension and used as template to run PCR for TK gene amplification. The amplified products were cloned into PC DNA3 vector and sequenced. The vector pcDNA/HSVⅡ TK was cut by endonuclease. The gained TK gene was cloned into eukaryon expression vector. pcDNA3/angiostation, which had been constructed. ResultsCoding region of HSVⅡTK gene consisted of 1 128 bp except stop code, it encoded 376 amino acids.After cutting the new vector by endonuclease Hind Ⅲ and BamH Ⅰ,we gained the following gene fragment: 1000 bp (TK) and 700 bp (angiostation).Conclusion The fusion gene eukaryon expression vector， pcDNA3/HSVⅡ TK/angiostatin has been constructed.
Objective To investigate the effect of topical treatment with antisense oligonucleotides（ASON）targeting tumor necrosis factor-alpha;（TNF-alpha;）on the pathological process of experimental herpes simplex virus type-Ⅰ（HSV-Ⅰ）induced chorioretinitis in mouse eye. Methods Fifty BALB/c mice were randomly divided into experimental and control group（twenty five mice in each group）.HSV chorioretinitis model was induced in each mouse by inoculating 1times;105 plaque-forming units （pfu） of HSV-Ⅰ（KOS strain）into anterior chamber of the right eye.In experimental group，Fluorescein isothiocyanate （FITC）-labeled ASON targeting TNF-alpha; 2 mu;l were injected sub-conjunctiva in the left eye1day before and 1 and 4 days after the infection;while phosphate buffer solution was injected in the same way in control group.The inflammation changes of the eyes in the 2 groups were observed and the clinical grades were assessed according to the extends of anterior-chamber inflammation，vasodilatation of cornea and iris，formation of cataract，and vitreous opacity. All of the mice were executed 10 days after the infection and were observed histologically. The contents of TNF-alpha; in retina and choroid were measured by enzyme-linked immunobsorbent assay（ELISA）. Results After the infection，acute inflammation appeared in the right eyes in both groups. The inflammation of the left eyes in experimental group was significantly milder than which in the control group.Twelve left eyes had necrotic chorioretinitis in different degrees in the control group while 2 left eyes had mild chorioretinitis in the experimental group. The difference of the number of inflammatory cells between the 2 groups was statistically significant in retina，choroid，and ciliary body（P＜0.05）and was not obvious in anterior chamber，vitreous cavity，and iris（P＞0.05）.The content of TNF-alpha; in choroid and retina was（60plusmn;1.25）pg in the experimental group and（305plusmn;1.03）pg in the control group（P＜0.05）. Conclusions TNF-alpha; ASON treating HSV-Ⅰinduced chorioretinitis may reduce the content of TNF-alpha; in affected mice eyes and decrease the inflammatory reaction. (Chin J Ocul Fundus Dis, 2006, 22: 245-248)
ObjectiveTo evaluate the therapeutic effect of liver transplantation (LT) combined with adenovirus-mediated delivery of herpes simplex virus thymidine kinase / ganciclovir (ADV-TK/GCV) in treatment of patients with hepatocellular carcinoma (HCC), so as to benefit more patients with HCC beyond the Milan criteria. MethodsThe clinicopathologic data of patients with HCC underwent LT by the author team since 2007 were collected and analyzed. The patients were assigned into simple LT group and LT+ADV-TK/GCV group. The 5-year cumulative overall survival rate and relapse free survival rate of all LT patients and the patients with LT beyond the Milan criteria by simple LT and LT+ADV-TK/GCV therapy were compared. Meanwhile, Cox regression was used to analyze the risk factors affecting long-term overall survival rate and relapse free survival rate of all patients with HCC after LT. ResultsA total of 216 patients eligible for inclusion were collected in this study, including 134 patients in the simple LT group and 82 patients in the LT+ADV-TK/GCV group, 162 of whom beyond the Milan criteria, including 101 patients underwent the simple LT and 61 patients underwent the LT+ADV-TK/GCV. There were no statistical differences in the baseline data between the simple LT and LT+ADV-TK/GCV in all patients and patients beyond the Milan criteria (P>0.05). There were no statistical differences in 5-year overall survival rate and relapse free survival rate of all patients with HCC (P>0.05). The 5-year cumulative overall survival rate of the LT+ADV-TK/GCV group was better than that of the simple LT group in the patients beyond the Milan criteria (χ2=4.11, P=0.047), but it was not found that the 5-year cumulative relapse free survival rate had statistical difference (27-month survival time as the critical value, P=0.46, P=0.06). Cox regression multivariate analysis results showed that the larger cumulative tumor diameter, the preoperative elevated serum alpha fetoprotein (>400 μg/L), later TNM stage, and without combination of ADV-TK/GCV therapy increased the probability of shorter overall survival of patients after LT; and the patient’s older age, the larger cumulative tumor diameter, and later TNM stage increased the probability of shorter relapse free survival after LT, and it was not found that the combination of ADV-TK/GCV therapy had an impact on the relapse free survival. ConclusionLT combined with ADV-TK/GCV therapy can obviously improve overall survival among patients beyond the Milan criteria, more patients with advanced HCC will be candidates for LT combined with ADV-TK/GCV therapy.
ObjectiveExploring the influencing factors of acute phase (≤ 21 days) seizures and epilepsy in patients with Herpes simplex virus encephalitis (HSE) in our hospital, including emergency and inpatient wards, mainly from the perspective of the number of mNGS of Herpes simplex virus (HSV) in cerebrospinal fluid. MethodsFrom January to Octomber 2023, 28 emergency and inpatient patients in our hospital were collected, excepted clinical datas were not detailed. In the end, 24 patients with HSE diagnosed were included in the study, and their clinical datas were collected, including age, gender, acute encephalitis syndrome, the form of the seizure and the number of seizures, time of lumbar puncture, and various indicators of cerebrospinal fluid [pressure, protein, cell count (mononuclear and multinucleated cells), metagenomic Next-generation sequencing (mNGS), neural autoantibodied associated with autoimmune encephalitis], electroencephalogram, cranial imaging examination reports, and treatment plans.ResultsA total of 24 patients were enrolled, including 9 patients with a history of hypertension and 4 patients with a history of diabetes. There were 18 males and 6 females, with an average age of (53.17±17.19) years. The maximum age was 73 years old, and the minimum age was 21 years old. Among the 24 patients, one patient first presented to the local hospital and then transferred to our hospital for lumbar puncture, so the time from the onset of the disease to lumbar puncture was 30 days, and the other patient’s family members refused to complete lumbar puncture at first,so the time of lumbar puncture was 14 days from the onset of the disease. The remaining 22 patients were all completed lumbar puncture within 7 days of onset, and all emergency patients completed lumbar puncture within 24 hours of admission to the Emergency Department. mNGS results: 23 cases were HSV-1, and the remaining 1 case was HSV- 2. There were a total of 6 cases of seizures and epilepy in the acute phase (≤21 days), with focal or generalized seizures or epileptic status as the main form, and 2 cases of seizures occurred. Among these 8 patients, 5 showed lesions in the frontal and temporal lobes on enhanced cranial MRI, while the other 3 showed no specific features. At the same time, mNGS of cerebrospinal fluid suggested that the sequence number of HSV was greater than 3 000 was related with seizures and epilepsy with acute HSE. Conclusion Seizures and epileysy in acute HSE were related with the presences of intracranial cortical involvement,and the number of viral sequences in mNGS was closely related to acute phase seizures and epilepsy.