Objective This study aims to construct and identify the chimeric antigen receptor NK92 (CAR-NK92) cells targeting NKG2D ligand (NKG2DL) (secreting IL-15Ra-IL-15) and verify the killing activity of NKG2D CAR-NK92 cells against multiple myeloma cells. Methods The extracellular segment of NKG2D was employed to connect 4-1BB and CD3Z, as well as IL-15Ra-IL-15 sequence to obtain a CAR expression framework. The lentivirus was packaged and transduced into NK92 cells to obtain NKG2D CAR-NK92 cells. The proliferation of NKG2D CAR-NK92 cells was detected by CCK-8 assay, IL-15Ra secretion was detected by ELISA and killing efficiency was detected by lactate dehydrogenase (LDH) assay. The molecular markers of NKp30, NKp44, NKp46, the ratio of apoptotic cell population, CD107a, and the secretion level of granzyme B and perforin were detected using flow cytometry. In addition, the cytotoxic mechanism of NKG2D CAR-NK92 cells on the tumor was verified by measuring the degranulation ability. Moreover, after NKG2D antibody inhibited effector cells and histamine inhibited tumor cells, LDH assay was utilized to detect the effect on cell-killing efficiency. Finally, the multiple myeloma tumor xenograft model was constructed to verify its anti-tumor activity in vivo. Results Lentiviral transduction significantly increased NKG2D expression in NK92 cells. Compared with NK92 cells, the proliferation ability of NKG2D CAR-NK92 cells was weaker. The early apoptotic cell population of NKG2D CAR-NK92 cells was less, and NKG2D CAR-NK92 cells had stronger cytotoxicity to multiple myeloma cells. Additionally, IL-15Ra secretion could be detected in its culture supernatant. NKp44 protein expression in NKG2D CAR-NK92 cells was clearly increased, demonstrating an enhanced activation level. Inhibition test revealed that the cytotoxicity of CAR-NK92 cells to MHC-I chain-related protein A (MICA) and MICB-positive tumor cells was more dependent on the interaction between NKG2D CAR and NKG2DL. After stimulating NKG2D CAR-NK92 cells with tumor cells, granzyme B and perforin expression increased, and NK cells obviously upregulated CD107α. Furthermore, multiple myeloma tumor xenograft model revealed that the tumors of mice treated with NKG2D CAR-NK92 cells were significantly reduced, and the cell therapy did not sensibly affect the weight of the mice. Conclusion A type of CAR-NK92 cell targeting NKG2DL (secreting IL-15Ra-IL-15) is successfully constructed, indicating the effective killing of multiple myeloid cells.
Humans ; Mice ; Animals ; Receptors, Chimeric Antigen/genetics* ; Interleukin-15 ; NK Cell Lectin-Like Receptor Subfamily K/metabolism* ; Granzymes ; Cell Line, Tumor ; Multiple Myeloma/therapy* ; Perforin

Gene therapy is a rapidly developing field. The most widely used technique for foreign gene transfer is lentiviral-mediated gene therapy. Lentiviral vector has been developed from the first generation to the third generation in terms of safety. The preparation of lentiviruses with high titer remains difficult. In this study, a Fibra-Cel sheet carrier was used as an HEK293T cell carrier matrix, and several sterile cell culture spinners were combined and cultured on a roller bottle machine to scale up the adherent cells. The virus titer was maximized by screening the factors to optimize the lentivirus titer in the third-generation lentivirus packaging process one by one. Fibra-Cel sheet vector was successfully used as the matrix of HEK293T cell adhesion to culture adherent cells at large scale. The optimal conditions for large-scale preparation of the third-generation lentivirus by bottle roller were screened and three batches of lentiviruses were produced on pilot scale. The production time of lentivirus was shortened from 120 hours to 54 hours from plasmid transfection to virus collection; in terms of cost, a rolling bottle machine was used instead of a bioreactor, leading to lower cost and no need for repeated sterilization during the whole process. The safe, effective and low-cost operation of successful production will provide a technical base for the large-scale preparation of lentivirus and thus lay a firm foundation for its clinical application.
Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; Transduction, Genetic ; Transfection

Objective: To investigate the clinical effect of percutaneous curved vertebroplasty in the treatment of thoracolum-bar osteoporotic vertebral compression fractures (OVCFs). Methods: All of 85 patients with single thoracolumbar vertebral OVCFs who met the admission criteria from January 2017 to July 2018 were divided into three groups according to the random dig-its table method. They were treated with percutaneous curved vertebroplasty, routine unipedicular PVP and routine bipedicular PVP respectively. There were 25 patients in the percutaneous curved vertebroplasty group, 6 males and 19 females; aged 56-80 years, with an average age of 70.6±9.7 years. Fracture vertebral body distribution: T₁₀ 2 cases, T₁₁ 4 cases, T₁₂ 3 cases, L₁ 9 cases, L₂ 3 cases, L₃ 1 case, L₄ 1 case and L₅ 2 cases. There were 32 patients in the routine unipedicular PVP group, 6 males and 26 fe-males; aged 58-75 years, with an average age of 69.5±9.3 years. Fracture vertebral body distribution: T₁₀ 2 cases, T₁₁ 4 cases, T₁₂ 5 cases, L₁ 11 cases, L₂ 6 cases, L₃ 1 case, L₄ 1 case and L₅ 2 cases. There were 28 patients in the routine bipedicular PVP group, 5 males and 23 females; aged 59-81 years, with an average age of 69.8±8.8 years. Fracture vertebral body distribution: T₁₀ 2 cases, T₁₁ 4 cases, T₁₂ 4 cases, L₁ 10 cases, L₂ 4 cases, L₃ 1 case, L₄ 1 case and L₅ 2 cases. The operation time, injected cement volume, in-traoperative blood loss were recorded and analyzed. Preoperative, postoperative 1 week and 3 months visual analogue scale scores and oswestry disability index were adopted to value the clinical improvements. Preoperative, postoperative 1 week and 3 months relative vertebral height and kyphosis correction, and the cement leakage rate were measured and analyzed. Results: There was no significant difference in the data of gender, age, VAS scores, ODI and distribution of fracture vertebrae among the three groups (P>0.05), and the baseline data was comparable. The average VAS score in the percutaneous curved vertebroplasty group was 2.3±0.5 at 1 week after surgery, that of the routine unipedicular PVP group was 2.4±0.4 and that of the routine bipe-dicular PVP group was 2.4±0.4; the average ODI in the percutaneous curved vertebroplasty group was 19.8%±3.9%, that of the routine unipedicular PVP group was 20.0%±4.1% and that of the routine bipedicular PVP group was 19.9%±3.8%; they were lower than the preoperative data, which were statistically significant (P<0.001). The average relative vertebral height in the percutaneous curved vertebroplasty group was 48.99%±9.23% at 3 months after surgery, that of the routine unipedicular PVP group was 47.11%±10.12% and that of the routine bipedicular PVP group was 46.71%±11.16%; the average kyphosis cor-rection in the percutaneous curved vertebroplasty group was 6.21%±1.94%, that of the routine unipedicular PVP group was 5.22%±2.07% and that of the routine bipedicular PVP group was 5.97%±2.09%; there was 1 cement leakage case in the per-cutaneous curved vertebroplasty group; those of the routine unipedicular PVP group were 4 cases and those of the routine bipe-dicular PVP group were 6 cases; there was no significant difference among the three groups (P>0.05). Operation time 39.10±2.00 min vs 38.70±1.70 min, injected cement volume 3.60±0.11 ml vs 3.50±0.13 ml and blood loss 5.10±0.30 ml vs 5.00±0.40 ml of the percutaneous curved vertebroplasty group and the routine unipedicular PVP group were less than those of the routine bipedicular PVP group, which were statistically significant (P<0.05). Conclution Percutaneous curved vertebroplasty could achieve satisfactory clinical outcomes for OVCFs, with advantages of less operation time, less blood loss, limited X-ray expo-sure, less injected cement volume, and more balanced augmentation for stabilization of the affected vertebrae and total verte-bral column.

Objective To establish a simple animal model of cough variant asthma(CVA)through sensitizing Brown-Norway(BN)rats with ovalbumin(OVA). Methods A total of 36 BN rats were randomly divided into three groups, including the normal control group,the model control group and the montelukast group. BN rats in the model group and the montelukast group were intraperitoneally administered with 2.0 mg of OVA and 100 mg of Al(OH)3,and the same volume of sterile saline was given to the normal group by intraperitoneal injection. Boosting was carried out by intraperitoneal administration with 0.01 mg of OVA and 100 mg of Al(OH)33 weeks later,and the rats in the normal group were injected with the same dose of physiological saline. Three weeks later,the actively sensitized BN rats were challenged with aerosolized OVA for 7 times on alternative days,and the rats in the normal group were treated with sterile saline instead of OVA. At the same time, the montelukast group was given 1.3 mg/kg of montelukast 30 minutes before atomization by intragastric administration once a day for 2 weeks,and the normal group and the model group were given the same volume of water. The tests of cough sensitivity to capsaicin and bronchial responsiveness were performed 24 h after the last administration. Results Compared with the normal group, the times of coughing(P< 0.01)and the lung resistance(RL)(P< 0.05)in the model group were significantly increased,while the lung compliance(Cdyn)was significantly decreased(P< 0.05). There was a significant difference(P < 0.05)in the times of coughing caused by capsaicin between the model group and the montelukast group. Compared with the model group,RLin the montelukast group was decreased significantly(P< 0.05), and Cdynwas increased significantly(P< 0.05). Conclusions This rat model of CVA is similar to a variety of clinical features of CVA and is easy to operate. Thus it can be used as an effective animal model of CVA.

Objective To explore the surgical efficacy of meningioma patients with epilepsy as the main symptoms. Methods A retrospective analysis of 124 meningioma patients with epilepsy as the main symptoms between January 2010 and January 2016,follow-up effect of epilepsy control. Results There were 94 cases of Simpson I orⅡgrade resection,27 cases of gradeⅢresection and 3 cases of grade Ⅳ resection. The surgical out-comes of patients were evaluated based on Engel′s classification. There were 98 cases(79.0%)of EngelⅠgrade, 11 cases(8.9%)of EngelⅡgrade,Engel Ⅲ and Ⅳ grade were 12 cases(9.7%)and 3 cases(2.4%). Among them,87 of 94 patients with Simpson I orⅡ grade resection epilepsy were well controlled,the satisfaction rate of epilepsy control was 92.6%. Conclusions Epilepsy as the main symptoms of meningioma patients need to clear about the seizure onset area preoperative,need to pay special attention to whether there is an independent epilepsy starting area of the distant site of the tumor. On the basis of the tumor resection as much as possible,at the same time dealing with the abnormal release of the cortex can effectively improve the efficacy of postoperative epilepsy control.

Objective: To research the anti-inflammatory and anti-allergic effects of Qifangbimin particle.Methods: The anti-inflammatory effects were observed by the methods of xylene-induced ear-swelling in mice and cotton-ball induced granuloma in rats.The anti-allergic effects were evaluated by the method of passive skin allergy model in rats and ear-heterogeneous passive skin allergy model in mice.Results: In the treatment groups with Qifangbimin particle, the swelling degree of ear edema induced by dimethylbenzene in mice was significantly suppressed when compared with that in the control groups (P<0.01 or 0.05) , however, the particle had no significantly inhibitory effect on granulation tissue hyperplasia induced by cotton-ball in rats.The Qifangbimin particle groups obviously decreased the absorbance value of locus coeruleus on rats' back (P<0.01 or 0.05), and Qifangbimin particle at high dose significantly reduced the absorbance value of locus coeruleus of auricle in mice (P<0.01 or 0.05).Conclusion: Qifangbimin particle has significant anti-inflammatory and anti-allergic effects.

Objective To explore the surgical treatment of epilepsy in rolandic and immediate perirolan dic cortex.Methods 376 patients accepted surgical treatment in our hospital from January 2010 to January 2016,of whom 48 patients fulfilled eligibility criteria and were included in the study.Patients were divided into 2 groups for analysis:patients with resections in the rolandic cortex (A group,n =28),and patients with resections in immediate perirolandic cortex and simultaneous sensorimotor multiple subpial transections (B group,n =20).Postoperative seizure outcome and neurologic deficits were analyzed.Results According to classification of Engel,Engel Ⅰ was in 22 (45.8％),Engel Ⅱ in 7 (14.6％),Engel Ⅲ in 10 (20.8％) and Engel Ⅳ in 9(18.8％).Seizure outcome satisfaction rate (Engel Ⅰ +Engel Ⅱ) in the group A was significantly higher than the group B (P =0.036);temporary and transient neurological deficits in the two groups had no statistically significant (P =0.569,P =0.418).Conclusions It is possible to achieve complete dissection of epileptic foci in rolandic and immediate perirolandic cortex without damage.The good prognosis is not related to the degree of excision of epilepsy and has no definite correlation with neurological dysfunction.

Objective To assess the efficacy of one-stage posterior median incision via costotransverse joint in treating thoracic spinal tuberculosis.Methods Thirty three patients with tuberculosis of thoracic spine undergoing one-stage posterior thoracic spine debridement,bone grafting fusion and posterior instrumentation from July 2011 to October 2013 were included in the study.There were 18 males and 15 females.The age was from 17 to 39 years old with an average of 29.5.The course was from 5 to 11 months with an average of 7.3.The Cobb angle was from 19° to 42° with an average of 29.8°.There were 5 in upper thoracic spine,17 in middle thoracic spine and 11 in lower thoracic spine.6 were in Frankel scale grade C,11 were in grade D and 16 were in grade E before surgery.Postoperative kyphosis correction,recovery of neurological function and bone fusion were observed.Results All the surgeries were completed successfully.The operation time was from 120 to 230 min with an average of 183.The blood loss during operation was from 480 to 700 ml with an average of 530.All 33 patients were followed-up for 12-36 months with an average of 22.7 months.The Cobb angle was from 8° to 15° with an average of 10.2°,the correction rate was 77％.The Frankel scale of 11 patients recovered from D to E,2 recovered from C to D and 4 recovered from C to E.The postoperative kyphosis correction and Frankel scale were significantly improved,all patients had a 100％ bone fusion rate and there were no internal fixation loosened or shift of graft bone at the last follow-up.ESR and CRP were returned to the normal range and the tuberculosis symptoms disappeared.Conclusions One-stage posterior median incision via costotransverse joint can complete surgery by the same position and the same incision in treating thoracic spinal tuberculosis with safety and good clinical efficacy.