Objective
To study effect of carcinoembryonic antigen (CEA) positive targeted lymphocytes on gastric cancer cells in vitro and in vivo.
Methods
The peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers. The recombinant vector anti-CEA-scFv-CD3ζ-pcDNA3.0 was transfected into the PBMCs by lipofectamine 2000, by this means, the CEA special lymphocytes were obtained. Meanwhile, the PBMCs transfected with empty plasmid pcDNA3.0 were used as control (empty vector lymphocytes). The different lymphocytes and gastric cancer cells (CEA positive KATOⅢ gastric cancer cells and CEA negative BGC-823 gastric cancer cells) were co-cultured, then the ability to identify the gastric cancer cells and it’s effect on apoptosis of gastric cancer cells were observed at 24 h or 36 h later respectively. The CEA special lymphocytes and empty vector lymphocytes were injected by the tail vein of nude mice bearing gastric cancer cells, then it’s effect on the tumor was observed.
Results
① The CEA special lymphocytes could strongly identify the KATOⅢ gastric cancer cells (identification rate was 72.3%), which could weakly identify the BGC-823 gastric cancer cells (identification rate was 7.8%). ② The apoptosis rate of the co-culture of CEA special lymphocytes and KATOⅢ gastric cancer cells was significantly higher than that of the co-culture of empty vector lymphocytes and KATOⅢ gastric cancer cells (P=0.032), which had no significant difference between the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells and the co-culture of empty vector lymphocytes and BGC-823 gastric cancer cells (P=0.118). ③ The tumor volume of the co-culture of CEA special lymphocytes and KATOⅢ gastric cancer cells was significantly smaller than that of the co-culture of empty vector lymphocytes and KATOⅢ gastric cancer cells (F=5.010, P<0.01) or the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells (F=4.982, P<0.01), which had no significant difference between the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells and the co-culture of empty vector lymphocytes and BGC-823 gastric cancer cells (F=1.210, P>0.05).
Conclusion
CEA special lymphocytes can promote cell apoptosis and inhabit tumor reproduction of CEA positive gastric cancer cells in vitro and in vivo.
Objective To construct a green fluorescent protein expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28, containing anti-TAG72 single chain variable fragment (scFv) fused into the transmembrane and intracellular domain of the signal-transducing chain of CD28 gene, and to transfect it into peripheral blood mononuclear cells． Methods Recombinant transmembrane and intracellular domain of CD28 cDNA and anti-TAG72 scFv cDNA fragment was subcloned into pEGFP-C3 vector. Recombinant clones were selected by Kanamyein， and then identified by PCR， enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into peripheral blood mononuclear cells by means of lipofection. The recombinant protein expression was confirmed by immunocytochemistry, laser scanning confocal microscope, PCR and Western blot analysis. Results The fused gene fragment of anti-TAG72 scFv-CD28 was successfully inserted into pEGFP-C3 plasmid, and it was confirmed by enzyme digestion and DNA sequencing. The fused anti-TAG72 scFv-CD28 gene and its protein was identified in peripheral blood mononuclear cells． Conclusion The eukaryotic expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28 was successfully constructed and transiently expressed in peripheral blood mononuclear cells， which would lay a foundation for further studies on the role of it to activate tumor-associated antigen-specific T lymphocyte, for generating of modified T lymphocytes targeting gastrointestinal tumors.
ObjectiveTo investigate the method for generating anchor chemric T lymphocytes that can target tumor associated glycoprotein-72 (TAG72) antigen and analyze their repressive effects on proliferation of TAG72 positive hepatocarcinoma cells. MethodsFirstly, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated. And then, CD8+ T cells were isolated from PBMCs via magnetic activated cell sorting (MACS). These lymphocytes were transfected with recombinant vector, anti-TAG72-scFv-CD28-pcDNA3, through Lipofectamine2000 to gernerate anchor chimeric TAG72-specific CD8+ T cells. SMMC7721 (TAG72 positive) hepatocarcinoma cells were co-cultured with chimeric T lymphocytes and their cell cycles were analyzed by flow cytometry (FCM). ResultsAnchor chmeric T lymphcytes targetting TAG72 recognized TAG72 positive SMM7721 cells and repressive effects on their proliferation were observed by flow cytometry. ConclusionAnchor chmeric T lymphcytes targetting TAG72 on tumor surface can specifically recognize TAG72 positive hepatocarcinoma cells and may exert repressive effect on their proliferation.
Objective To evaluate the analgesic effect of intra-articular ropivacaine with lidocaine. Methods A double-blind randomized controlled trial was conducted. Ninety patients receiving selective knee arthroscopy were randomized into three groups of 30 patients. At the end of the operation, before the release of the tourniquet, an intra-articular injection was administered to each patient through arthroscope, in accordance with their random allocation: 0.9% normal saline (normal saline group); 100 mg ropivacaine (ropivacaine alone group) and 100 mg ropivacaine and 100 mg 2% lidocaine (ropivacaine with lidocaine group). Pain intensity was assessed after the operation using the 100-mm visual-analog scale (VAS), and the amount of supplemental analgesics used within the following 24 hours were recorded. Results The VAS scores of 2 hours postoperatively at rest, and 1, 2, 4, and 8 hours postoperatively at motion, were significantly higher in the normal saline group than in ropivacaine alone group (Plt;0.05). The VAS scores 0.5, 1 and 2 hours postoperatively at rest, and at the awaking moment, 0.5, 1, 4, 8, and 24 hours postoperatively at motion, were significantly higher in the normal saline group than in ropivacaine with lidocaine group (Plt;0.05). Conclusion Intra-articular ropivacaine can reduce a patient’s pain after operation. The combination of lidocaine with intra-articular ropivacaine can reduce the patient’s pain severity immediately after the operation and achieve an early analgesic effect.
Objective To discuss left ventricular reconstruction methods and effectivness in patients with left ventricular aneurysm after myocardial infarction. Methods Between June 2003 and August 2008, 23 patients with left ventricular aneurysm after myocardial infarction were treated. Of them, 13 were male and 10 were female with an average age of 61.2 years (range, 47-74 years). According to New York Heart Association (NYHA) criteria for cardiac function, there were 3 cases of grade I, 6 cases of grade II, 10 cases of grade III, and 4 cases of grade IV. The coronary arteriongraphy showed singlevesseldisease in 2 cases, double-vessel disease in 5 cases, triple-vessel disease in 16 cases. The locations of ventricular aneurysm were the apex cordis in 18 cases, antetheca and parieslateral is in 4 cases, and interior wall in 1 case. The left ventricular ejection fraction was 36.52%± 12.15%, and left ventricular diastol ic final diameter was (62.30 ± 6.52 ) mm. Nine patients received standard l inear repair, 6 patients received standard l inear repair after endocardial ring shrinkage, and 8 patients received patch suture after endocardial ring shinkage. Results Two cases died perioperatively, and re-thoracotomy was performed to stop bleeding in 1 case. Incisions healed by first intention in the other patients without early compl ication. Twentyone patients were followed up 7-48 months (median, 19 months). At 6 months after surgery, the left ventricular ejection fraction 46.52% ± 9.41% were significanly improved when compared with that at preoperation (t=2.240, P=0.023); the left ventricular diastol ic final diameter (52.23 ± 5.11) mm were significantly decreased when compared with that at preoperation (t=2.170, P=0.035). The cardiac function according to NYHA criteria was at grade I in 8 cases and at grade II in 13 cases. One patient died of cerebral hemorrhage at 18 months after operation and the activities of daily l iving recoverd in the others. Conclusion Individual theraputic methods are used according to patients’ different conditions for left ventricular aneurysm after myocardial infarction.
