[Development of oral vaccine carrying GCPII gene and its role in reducing the dosage of pentobarbital in rat: a primitive research].

OBJECTIVE: To develop an oral vaccine carrying glutamate carboxypeptidase II (GCP II) and to explore whether it can affect the dosage of pentobarbiturate. METHODS: Polymerase chain reaction, digestion of endonuclease and ligation, blue-white selection were used to construct an expression vector pcDNA3.1-GCP II. HEK293 cells were cultured. The vector pcDNA3.1-GCP II was transfected into the HEK293 cells by Ca(3)(PO(4))(2) coprecipitation method. The transfected HEK293 cells were cultured in HEM liquid culture prepared with G418. Three weeks after, positive clones, HEK293-GCP II, were identified. Reverse-transcription PCR and immunofluorescence cell staining were used to testify positive cell line; Method of CaCl(2) was used to prepare oral vaccine of attenuated Salmonella typhimurium carrying GCP II (SL-GCP II). Expression of SL-GCP II in vitro was observed by adding SL-GCP II into the primarily cultured macrophage. Fifty male SD rats were randomly divided into 2 groups of 25 rats: group A, undergoing intragastrical infusion of SL-GCP II, 600 micro l/time, in total 4 times in 4 days; and group B, as control group, undergoing intragastrical infusion of SL3261. Fifteen days after, 5 g/L pentobarbital sodium was injected intraperitoneally with the first dosage of 1.0 ml and the response was observed in 10 minutes, then 0.1 ml was added every time. The specific dosage of pentobarbital sodium was recorded when anesthesia meeting the requirement of operation was reached. Phenobarbital sodium of this dosage was used to anesthetize the rats to observe the response of the rats. Immunofluorescence method was used to detect the titer of antibody in rat circulation with HEK293 GCP II cells as target cells. RESULTS: An expression vector containing GCP II, pCMV-GCP II, pCDNA3.1-GCP II was constructed. The cell line, HEK 293-GCP II was established. In vitro experiment proved that primarily cultured macrophage phagocytized SL-GCP II and effectively expressed GCP II gene. After infusion of the oral vaccine 22 of the 25 SD rats of the group A produce GCP II antibodies. The dosage of pentobarbiturate used in experimental group was 36.9 mg/kg +/- 1.6 mg/kg; significantly lower than that in the control group (40.8 mg/kg +/- 1.4 mg/kg, P = 0.00). CONCLUSION: An oral vaccine carrying GCP II gene has been developed that activates the immune response of rat to produce GCP II antibodies and lower the dosage of pentobarbiturate needed.

[Experimental study on the proliferation and plasticity of neural stem cells in situ in adult rats after cerebral infarction].

OBJECTIVE: To investigate the proliferation and plasticity of neural stem cells in situ in adult rats after cerebral infarction. METHODS: Cerebral infarction models of rats were made and the dynamic expression of bromodeoxyuridine (BrdU) and BrdU/polysialylated neural cell adhesion molecule (PSA-NCAM) were determined by immunohistochemistry and immunofluorescence staining. RESULTS: Compared with the controls, the number of BrdU-positive cells in the subventricular zone (SVZ) and hippocampus increased strikingly at day 1 (P < 0.05), reached maximum at day 7, and decreased markedly at day 14, but it was still elevated compared with that of the controls (P < 0.05); The number of BrdU-labeled with PSA-NCAM-positive cells increased strikingly at day 7 (P < 0.05), reached maximum at day 14, and markedly decreased at day 28, but it was still elevated compared with that of the controls (P < 0.05), and was equal to 60% of the number of BrdU-positive cells in the same period. CONCLUSIONS: Our results indicate that cerebral infarction stimulate the proliferation of inherent neural stem cells in situ and most proliferated neural stem cells represent neural plasticity.

[The experimental study of rAAV2 transfecting neural stem cells-derived neurospheres].

AIM: To investigate the abilities of recombinant adeno-associated virus type 2 (rAAV2) transfecting neurospheres. METHODS: The rAAV2 conjugated with FITC (rAAV2-FITC) was added into the culture medium of neurospheres and 30 minutes later the neurospheres were detected with a fluorescence microscopy to determine if the AAV can combine with neurospheres. The rAAV2 containing GFP reporter gene (rAAV2-GFP) was incubated with the neurospheres for a month and then detected the ability of transfecting neurospheres. The neurospheres transfected with rAAV2-containing GFP were transplanted to the brain of rats. A month later the rats were sacrificed and the brains were removed to detect if there are expressions of the reporter gene. The neurospheres were transfected with rAAV2 containing hypoxia responds elements (HRE) and vascular endothelium growth factor(VEGF) gene and reporter gene GFP (rAAV2-HRE-VEGF-GFP) and then cultured in low oxygen density environments. Seventy-two hours later the neurospheres were detected through a fluorescence microscopy. RESULTS: The neurospheres incubated with rAAV2-FITC present bright green fluorescence. GFP, the reporter gene, can be seen clearly 1 month after being transfected with rAAV2-GFP. The same green fluorescence protein can be observed ex vivo as well. The fluorescence can be seen in neurospheres transfected by rAAV2-HREVEGF-GFP only in low oxygen density. CONCLUSION: The rAAV2 can transfect neurospheres specifically and efficiently. Reporter gene can be expressed in the neurospheres in vivo and ex vivo. Expression of reporter gene can be adjusted by HRE.

[Production and purification of helper free recombinant adeno-associated virus 2].

OBJECTIVE: To explore an efficient and simple way to product and purify helper free adeno-associated virus vectors. METHOD: Helper free rAAV system was transferred into HEK293T cells through phosphorated calcium method, thus producing rAAV, then the rAAV vector was purified through chloroform-PEG8000/NaCl-chloroform method and ultrafiltration. SDS-PAGE protein electrophoresis and Western blotting were used to detect the rAAV protein. The quantity of rAAV genome was determined through blot hybridization. HEK293T cells were cultured, rAAV was added, and fluorescence microscopy was used to count the amount of cells expressing green fluorescent protein so as to measure the transferring unit of rAAV. RESULTS: rAAV2 was thus produced with a particle number of 2 x 10(13)/ml and a transferring unit of 5 x 10(11)/ml. CONCLUSION: This method to produce and purify rAAV is efficient and simple, without need of any special equipment, and can be finished in a common laboratory.

[Proliferation and differentiation of neural stem cells after cerebral infarction: an experimental study of adult rats].

OBJECTIVE: To investigate the proliferation and differentiation of neural stem cells after cerebral infarction(CI) in adult rats. METHODS: CI animal model was made by ligating the common carotid artery and external carotid artery and inserting a piece of nylon thread into the internal carotid artery among 100 male Wistar rats. Then the rats were randomly divided into 5 groups: group of I day after brain infarction (n = 20), group of 3 days after brain infarction (n = 20), group of 7 days after brain infarction (n = 20), group of 14 days after brain infarction (n = 20), and group of 28 days after brain infarction (n = 20). Twelve rats undergoing sham operation with a piece of nylon thread inserted only into the common carotid artery were used as controls. The rats were killed at different time points and their brains were taken out. The expression of bromodeoxyuridine (BrdU) and Musashil (both used to mark the dividing neural stem cells), and of glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen (NeuN) (both used to mark the differentiating neural stem cells) were determined by immunohistochemistry and immunofluorescence staining. RESULTS: In the normal brain tissues, only a small amount of BrdU(+) cells were found in the hippocampus. One day after CI the number of BrdU(+) cells began to increase in the hippocampus at the CI side (P < 0.05), peaked 7 days after CI with a number 6 times that at the normal side, began to decrease 14 days after, and almost reached normal 28 days after. The number of BrdU(+)/Musashil(+) cells began to increase 1 day after CI (P < 0.05), peaked 7 days after, began to decrease 14 days after, and almost reached normal 28 days after. The number of BrdU(+)/GFAP(+) cells at the CI side remained almost unchanged after CI. The number of BrdU(+)/NeuN(+) cells began to increase 14 days after CI (P < 0.05) and peaked 38 days after. CONCLUSION: Cerebral infarction stimulates the proliferation of inherent neural stem cells and most proliferated neural stem cells differentiate into neurons.

[Measurement of soluble CD44v6 in peripheral blood as assistant diagnosis of invasive pituitary adenomas].

OBJECTIVE: To investigate the value of measuring the concentration of soluble CD44 splice variant 6 (sCD44v6) in peripheral blood in patients with invasive and non-invasive pituitary adenomas. METHODS: The concentrations of sCD44v6 in peripheral blood were measured with ELISA in 68 patients with invasive pituitary adenomas and 100 patients with non-invasive pituitary adenomas. RESULTS: The serum concentration of sCD44v6 in patients with invasive pituitary adenomas was lower than that in patients with non-invasive pituitary adenomas, while the latter was lower than that in healthy controls. The serum concentrations of sCD44v6 were (44.63 +/- 7.21), (34.53 +/- 6.41), and (26.34 +/- 4.95) ng/ml in patients with invasive microadenoma, macroadenoma, and giant adenoma, and (60.78 +/- 9.61), (57.78 +/- 10.00), and (37.22 +/- 5.17) ng/ml in patients with non-invasive microadenoma, macroadenoma, and giant adenoma, lower than that in the healthy control group (68.73 +/- 6.00) ng/ml. Significant differences were observed among groups (P < 0.005). The concentration of sCD44v6 in peripheral blood decreased as the tumor size increased (P < 0.01), which was particularly significant in invasive pituitary adenomas. The positive rate in the patients with invasive pituitary adenomas reached 89.71%. CONCLUSION: Serum concentration of sCD44v6 in the peripheral blood is inversely correlated with tumor size and its invasive growth, which may provide certain value in the early diagnosis, treatment and prognosis of invasive pituitary macroadenoma and giant adenoma.