[Construction of shRNA lentivirus vector on rat DREAM gene and its analgesic effect on CCI rats].
OBJECTIVE
To construct the recombinant lentivirus vector containing short hairpin RNA (shRNA) inhibited DREAM expression and to investigate the gene therapy of neuropathic pain by inhibiting the expression of DREAM gene by RNA interference.


METHODS
An effective short hairpin RNA targeting to rat DREAM was cloned into the plasmids on the base of Lentivirous vectors, pKCSHR-Puro/GFP, and both of the pKCSHR-Puro/GFP-DREAM and Lentivector package plasmids mix were transferred into the 293T cells. The culture supernatant was harvested, and the virus titer was detected 48 hours after transferring. Thirty-six sheer breed pathogen free adult Sprague Dawley rats were randomly divided into 6 groups (6 in each group): normal control group (N); sham-operated group (S); CCI group (C0 group):CCI model without any intervention; Saline control group (C1 group); empty vector control group (C2 group); and LV-shRNADREAM lentiviral vector treatment group (C3 group). The rats in the last 3 groups respectively accepted injection of normal saline, blank vector, LV-shRNADREAM lentiviral vector in the subarachnoid on the 7th day after CCI, and the pain behavior was observed after 3, 7, 10, 14, 21 d after CCI. Green fluorescent protein (GFP) expression was detected by fluorescence microscope and the contents of DREAM mRNA and DREAM protein were detected by Realtime PCR and Western blot respectively in the rat lumbar spinal cord.


RESULTS
The short hairpin RNA sequences targeting at rat DREAM were cloned into the vectors, and an entry clone and an expression clone were constructed successfully confirmed by sequence analysis. Lentiviral packaging was successful in 293 T cell line and the transfection titer of the lentivirus was 1 x 10(8) IFU/mL. LV-shRNADREAM lentivirus vector was transfected successfully in the rat spine with Intrathecal injection of LV-shRNADREAM. Compared with the other 3 groups, heat pain threshold and mechanical pain threshold in Group C3 improved significantly (P<0.01), and the expression of DREAM mRNA and DREAM protein in the lumbar spinal cord in Group C3 were lowered significantly (P<0.01).


CONCLUSIONS
Lentivirus vectors containing rat DREAM gene are constructed successfully, and lentivirus mediated shRNA can inhibit the DREAM expression in the rat spine, which may prove to be an effective method for neuropathic pain.
Wang
Yunjiao
Y
Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha 410008, China.
Cheng
Zhigang
Z
Yu
Peng
P
Li
Jingyi
J
Bai
Nianyue
N
He
Zhenghua
Z
Yang
Shenghui
S
Guo
Qulian
Q
D000698:Analgesia / Q000379:methods*; D000818:Animals; D001483:Base Sequence; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D051605:Kv Channel-Interacting Proteins / Q000096:biosynthesis / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010146:Pain / Q000209:etiology; D059408:Pain Management; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics*; D012584:Sciatic Nerve / Q000293:injuries*; D014162:Transfection
C496760:Kcnip3 protein, rat; D051605:Kv Channel-Interacting Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins