ABSTRACT
Objective To evaluate the release of leucine-enkephalin (L-EK) in the cerebrospinal fluid induced by intrathecal human embryonic kidney (HEK293) cells modified with human preproenkephalin (hPPE)gene and the analgesic efficacy of L-EK for bone cancer pain.Methods Forty CIBP female Sprague-Dawley rats were randomized into transplantation (CIBP+ hPPE/HEK293,n =20) and control (CIBP + HEK293,n =20)groups using a random number table.At 1 day before inoculation of cancer cells (T1,baseline) and 8,15,21,25,32 and 35 days after inoculation (T2-7),thermal paw withdrawal latency (TWL) was measured,and the number of licking/biting the claw on the transplantated side and degree of hindlimb limping during free activities were recorded.After observation at T4,10 rats were chosen from each group and sacrificed and the cerebrospinal fluid of rats was collected in an ice bath for detection of hPPE expression using radioimmunoassay.Results Compared with control group,TWL was significantly prolonged,the concentration of L-EK in the cerebrospinal fluid was increased,and the number of licking/biting the claw on the transplantated side and degree of hindlimb limping during free activities were decreased at T4-7 in transplantation group.Conclusion Intrathecal HEK293 cells modified with hPPE gene can continuously secrete L-EK and mitigate bone cancer pain.
ABSTRACT
ObjectiveTo construct human embryonic kidney cells (HEK293) modified with human preproenkephalin (hPPE) gene.MethodshPPE gene fragments were obtained from recombinant plasmid pcDNA3.1( + )/hPPE by using restriction endonuelease Hind Ⅲ and Not Ⅰ.Homologous recombination of lentivirus and hPPE gene was produced by using recombinant DNA technology.HEK293 cells were then transfected with the recombinant lentivirus vectors.The expression of hPPE gene in HEK293 cells was detected by Western blot.ResultsThe results of DNA sequencing indicated that the positive clone of recombinant lentivirus was completely consistent with sequencing of hPPE in Genebank.The titer of the concentrated virus was 2.07 × 108 TU/ml.GFP fluorescence was not seen in HEK293 cells transfected with the lentiviral vector under fluorescence microscope.A strong fluorescence was seen in HEK293 cells transfected with Ubc-GFP-L.V.empty viral vector.Positive expression of hPPE was demonstrated in HEK293 cells transfected with lentiviral vector by Western blot.Conclusion HEK293 cells modified with hPPE gene were successfully constructed and the target gene hPPE was stably expressed in HEK293 cells.
ABSTRACT
Objective To establish a tibial cancer pain model with MADB-106 mammary gland carcinoma cell line and to conduct therapeutic research through the behavior pain, X-ray, histological observation of the model. MethodsA rat model of bone cancer pain was established by intra-tibial inoculations of MADB-106 rat mamnary gland carcinoma cells in SD rats. Spontaneous pain was assessed by the reflection of spontaneous paw withdraw, move-evoked pain was observed by the extent of lower extremity claudication when the rats walked and heat hyperalgesia was evaluated by using a thermal dolorimeter. The structural damage of the bone was monitored by X-ray and histology.ResultsThe model group spontaneously withdrawed their paws (14.42±1.24) times on the 15th day after operation (P ＜0.001), (18.33±1.37) times on the 22nd day (P ＜0.001), (21.25±1.54) times on the 25th day (P ＜0.001). The radiant pain thresholds of the model group was (1 1.86±1.63) s on the 15th day after operation (P ＜0.001), (8.38±1.05) s on the 22nd day (P ＜0.001), (7.47±1.25) times on the 25th day (P ＜0.001). The move-evoked pain score of the model group was (1.25±0.62) on the 15th day after operation (P ＜0.001), (2.00±0.95) on the 22nd day (P ＜0.001), (2.33±1.07)on the 25th day (P ＜0.001). The results showed that rats of the model group displayed the gradual development of spontaneous pain, heat hyperalgesia and move-evoked pain on the 15-25 days after injection of the tumor cells. The X-ray of the tibia showed clear bone destruction. The histology of the bone showed the bone marrow cavity was full of tumor cells and the cortical bone had been destroyed. ConclusionThe bone cancer model has been built well and it will be useful to evaluate the therapy of cancer pain after two weeks.