Study on the expression levels of CXCR4, CXCL12, CD44, and CD147 and their potential correlation with invasive behaviors of pituitary adenomas.

OBJECTIVE: To evaluate the factors of CXCR4, CXCL12, CD44, and CD147 as early potential diagnostic biomarkers by determining their expression levels in invasive and non-invasive pituitary adenomas. METHODS: Fresh pituitary adenoma specimens were collected from 35 pituitary adenoma (21 invasive and 14 non-invasive) patients who underwent surgical treatment in our Neurosurgery Department between January and April of 2009. The expression levels of CXCR4, CXCL12, CD44, and CD147 were evaluated firstly by flow cytometry, fluorescence microscopy in single cell suspensions, and then by immunohistochemical staining of paraffin tissue sections. RESULTS: Flow cytometric analyses showed that the percentage of CXCR4- and CXCL12-positive cells from invasive pituitary adenomas (IPA) was significantly higher in the single cell suspensions than that from non-invasive pituitary adenomas (nIPA) (P<0.05). Immunohistochemical staining revealed that CXCR4 and CXCL12 staining index scores of the invasive pituitary adenomas were significantly higher than those of the non-invasive pituitary adenomas (P<0.05). In contrast, neither flow cytometry nor immunohistochemical staining demonstrated significant difference between CD44 and CD147 expression levels, respectively. CONCLUSION: Expression levels of CXCR4 and CXCL12 are correlated with the invasiveness of pituitary adenomas. Therefore, rather than CD44 and CD147, CXCR4 and CXCL12 may potentially serve as biomarkers for early detection of pituitary adenomas.

Postacute ischemia vascular endothelial growth factor transfer by transferrin-targeted liposomes attenuates ischemic brain injury after experimental stroke in rats.

Our objective was to achieve the enhanced delivery of vascular endothelial growth factor (VEGF) to ischemically disordered brain through transferrin-coupled liposomes (Tf-PLs) via intravenous administration, and to observe the effect of Tf-VEGF-PLs on ischemic brain neuroprotection and angiogenesis. Cerebral VEGF overexpression was achieved with Tf-PLs by intravenous injection 48 hr after an acute stroke. ß-Galactosidase expression was monitored; saline was injected as a control. The success of postischemic gene transduction was confirmed by ß-galactosidase staining and by increased VEGF mRNA and protein in ischemic brain. Vascular density, neurological recovery, and ischemic area calculation were performed to evaluate the effect of Tf-VEGF-PLs. The positive expression of ß-galactosidase indirectly indicated that VEGF was successfully delivered into brain by Tf-VEGF-PLs. VEGF mRNA in the Tf-VEGF-PL group 24 hr after injection was significantly higher than in the control group (p < 0.05). Western blot analysis showed that postischemic Tf-VEGF-PLs resulted in increased VEGF protein levels compared with VEGF-PLs and saline-administered rats (p < 0.05) 48 hr after administration. At 21 days after drug injection, we observed a significant decrease in infarct volume and better neurological function in the Tf-VEGF-PL-treated group, compared with the VEGF-PL group. FITC-dextran marking showed increased vascular density in the penumbra of Tf-VEGF-PL-treated hemispheres (245,873.9, number of microvessels per field) compared with that in VEGF-PL-treated hemispheres (139,801.3) or saline-treated hemispheres (102,175.5) (p < 0.05). The remainder of the cerebral blood flow after ischemia in the Tf-VEGF-PL group was significantly more than in the control groups (0.35 vs. 0.29, 0.21; p < 0.05). We conclude that the VEGF gene can be delivered noninvasively into the brain by Tf-VEGF-PLs. Postischemic treatment with Tf-VEGF-PLs effectively promoted neuroprotection and vascular regeneration in the chronic stage of cerebral infarction.

[Preparation of brain targeted immunoliposomes].

To prepare a kind of effective non-viral transduction vector, which can deliver exogenous gene into the brain, this vector can be injected through vein system and has the ability to penetrate blood brain barrier. Several groups of materials proportion, type of oil phase, water-oil ratio, phosphatides-cholesterol ratio, temperature of steaming, ultrasonic temperature and time were compared for optimization. Well-constructed immunoliposomes encapsuling LacZ gene were infused into rats through tail vein. 48 h after injection, expression product beta-galactosidase of LacZ gene was detected by histochemistry staining to convince the validity of immunoliposomes as non-viral vectors. The best proportion of synthesis immunoliposomes is as following: phosphatides-cholesterol ratio is 1:1, lipids/drug is 100:1, the type of oil phrase is dichloromethane, oil-water ratio is 4:1, temperature of steaming is 30 degrees C, ultrasonic temperature and time is 10 degrees C and 5 min. At last, 10% trehalose was added as a stabilizer. The entrapment rate is 87.24% and antibody coupling rate is 69%. When immunoliposomes were infused into rats, the expression of LacZ gene could be observed in the brain and periphery organs. Through the best proportion of materials, gene delivering immunoliposomes had been synthesized successfully. This non-viral vector can deliver exogenous gene penetrating blood brain barrier and express in the brain, and will be well-used in the field of gene therapy of cerebral diseases.

[Specific expression in brain of exogenous gene by transpassing blood-brain barrier after intravenous injection].

OBJECTIVE: To study the expression of exogenous LacZ gene in brain via a delivery of OX26-pGFAP-IL. METHODS: pCMV-Liposome, OX26-pCMV-IL, OX26-pGFAP-IL and blank liposome were injected into rats via femoral vein. At 24 h post-injection, the method of Q-PCR was adopted to calculate the relative quantities of LacZ gene mRNA in brain and peripheral organs. At 48 h post-injection, the protein expression of LacZ gene was detected by the activity of beta-galactosidase and the method of histochemical stain. RESULTS: The result of Q-PCR showed that, at 24 h post-injection, the relative quantities of LacZ mRNA in OX26-pCMV-IL group (49.2 x 10(-6)) and OX26-pGFAP-IL group (44.9 x 10(-6)) were significantly higher than pCMV-liposome and blank liposome groups (P < 0.05). In peripheral organs, the relative quantity of LacZ mRNA in OX26-pCMV-IL group were significantly higher than that in OX26-pGFAP-IL group (P < 0.05). At 48 h post-injection, the activity of beta-galactosidase in OX26-pCMV-IL (0.67 pg/mg) and OX26-pGFAP-IL groups (0.92 pg/mg) were significantly higher than pCMV-liposome and blank liposome groups (P < 0.05). There was no significant difference between OX26-pCMV-IL group and OX26-pGFAP-IL group in terms of the expression of beta-galactosidase. The result of histochemical stain showed that OX26-pGFAP-IL achieved a specifically positive expression in brain and had a decreased expression in peripheral organs. CONCLUSION: OX26-pGFAP-IL injected via femoral vein can cross the brain-blood barrier and achieve a specific expression in brain under the control of GFAP promoter. OX26-pGFAP-IL decreases the non-specific expression in peripheral organs and it may be used as an non-viral gene therapy for intra-cranial diseases.

[Ferumoxide labeled Flk1+ CD31- CD34- human bone marrow mesenchymal stem cells and its in vivo tracing in the brains of Macaca Fascicularis].

OBJECTIVE: To explore the method for labeling Flk1+ CD31- CD34- human bone marrow mesenchymal stem cells (hBMSCs) with ferumoxide-PLL and evaluate the feasibility of its tracing after transplantation into the brains of Macaca Fascicularis. METHODS: The hBMSCs were incubated with ferumoxide-PLL. Trypan blue staining, Prussian blue staining, and transmission electron microscope were performed to show intracellular iron, marking efficiency, and the vigor of the labeled cells. After the hBMSCs were transplanted into the brains of cynomolgus monkeys by stereotaxis, magnetic resonance imaging (MRI) was performed to trace the cells in vivo. Cell survival and differentiation were studied with immunohistochemistry, Prussian blue staining, and HE staining. RESULTS: The marking efficiency of the ferumoxide-PLL was 96%. Iron particles were found intracytoplasmic of the hBMSCs by Prussian blue staining and transmission electron microscopy. The relaxation rates of labeled cells in MRI were 4.4 and 4.2 times higher than those of the unlabeled cells. Hypointensity area was found by MRI three weeks after transplantation. Many hBMSCs and new vessels were found in the transplantation zone by pathological and immunofluorescence methods. CONCLUSIONS: Ferumoxide-PLL can effectively label hBMSCs and thus increase its contrast in MRI results. The cells can survive in the brains of cynomolgus monkeys. The labeled hBMSCs can be traced in vivo by MRI.

Transplantation of neural stem cells modified by human neurotrophin-3 promotes functional recovery after transient focal cerebral ischemia in rats.

The study tested the hypothesis that transplantation of human neurotrophin-3 (hNT-3) over-expressing neural stem cells (NSCs) into rat striatum after a severe focal ischemia would promote functional recovery. Rat NSCs, transduced by Flag-tagged hNT-3 gene mediated by lentiviral vector (LV), were transplanted into the striatum ipsilateral to the injury of adult rats 7 days after 2-h occlusion of the middle cerebral artery (MCAO). From 3 days to 2 weeks after transplantation, the modified cells (NSCs-hNT3, as defined by Flag immunofluorencence staining) that survived the transplantation procedures could secrete significantly higher levels of neurotrophin-3 protein in the graft sites than controls (P<0.001). Furthermore, the rats that accepted NSCs-hNT3 exhibited enhanced functional recovery on neurological and behavioral tests, compared with controlled animals transplanted with saline or untransduced NSCs. This study suggests: (1) LV is an ideal vector to transduce foreign gene into the NSCs; (2) modified NSCs could carry therapeutic genes to disease tissues and express effectively; (3) modified cells could survive in the ischemic brains and continue to secrete neurotrophin-3 abundantly for over 2 weeks, which might have values for enhancing functional recovery after stroke.

[Treatment of stroke in rats with bone marrow mesenchymal stem cells].

OBJECTIVE: To investigate the effects of treatment of stroke in rats with bone marrow mesenchymal stem cells (BMSCs) and mechanism thereof. METHODS: Bone marrow of a healthy volunteer was collected and the BMSCs were separated with density gradient centrifugation. The hBMSC were cultivated and harvested until the third passage. A number of adult male Sprague-Dawley rats received corresponding behavioral training before surgery and underwent transient middle cerebral arterial occlusion (MCAO) for 2 hours. Sixty of them showing the scores of 6 approximately 12 according to the modified neurological severity score system were randomly divided into 2 groups: treatment group (n = 48, injected into the cortex around the ischemic areas with hBMSCs 3x10(5)/15 microl) and control group (n = 12, injected with D-Hanks solution 15 microl 24 hours after the establishment of MCAO models. Morris water maze test, Rotarod test and adhesive-removal test were performed since the 4th day to the 32 day after transplantation once every 3 days. 1, 2, 3, and 4 weeks after the transplantation 12 rats from each group were killed randomly to take out their brains. Immunofluorescence was used to identify the migration, survival and differentiation of the hBMSC. RESULTS: A large number of hBMSC could be seen within 2 weeks after transplantation. The number of hBMSC decreased since the 21st day after transplantation and few cells could be found at the end of 1 month after. No definite evidence supported the differentiation of neural cells derived from the hBMSCs during the whole process. Morris water maze test showed that the mean escape time 1 week after transplantation of the treatment group was (69 +/- 10) s, significantly shorter than that of the control group [(120 +/- 0) s, P < 0.05] The significant difference persisted until the 4(th) week (P > 0.05). Rotarod test with the speed of 10 r/min showed that the mean latency period 10 days after transplantation of the treatment group was (167 +/- 18) s, significantly longer than that of the control group [(37 +/- 19) s, P < 0.05]. The significant difference persisted until the experimental terminal. The adhesive-removal test showed that the mean latency period 13 days after transplantation of the treatment group was (33 +/- 8) s, significant shorter than that of the control group [(84 +/- 13) s, P < 0.05]. The significant difference persisted until the experimental terminal. CONCLUSION: Injection of hBMSCs into brain cortex improves neurological functional recovery after stroke. The transplanted cells can migrate and survive for a certain period, but no hBMSC express proteins phenotype of neural cells.

[In vivo tracking of bone marrow mesenchymal stem cells labeled with superparamagnetic iron oxide after cerebral ischemia in rats by magnetic resonance imaging].

OBJECTIVE: To explore the feasibility of in vivo tracking of bone marrow mesenchymal stem cells (BMSCs) labeled with superparamagnetic iron oxide (SPIO) by magnetic resonance imaging (MRI) in rats after cerebral ischemia, and to analyze the influence of stem cell therapy on the volume of cerebral infarction. METHODS: The samples of rat bone marrow were collected. BMSCs separated by density gradient centrifugation were cultivated and harvested until the third passage. BMSCs were labeled with SPIO, which was mixed with poly-L-lysine. The labeling efficiency was evaluated by Prussian blue staining. Transient middle cerebral arterial occlusion (MCAO) was performed successfully in 18 adult Sprague-Dawley rats that scored from 6 to 12 by the modified neurological severity test. The 18 rats were then randomly divided into group A, B, and C, with 6 rats in each group and Group C was regarded as control group. BMSCs were injected into the contralateral cortex of ischemia in group A, ipsilateral corpora striata in group B, while D-Hank's solution was injected into ipsilateral corpora striata (group C) 24 hours after MCAO. MRI was performed 1 day after MCAO, 1 day and 14 days after transplantation. The volume of infarcted brain tissue was measured and analyzed. Prussian blue staining of brain tissues was performed to identify the migration of BMSCs. RESULTS: The labeling efficiency of BMSCs with SPIO was 96%. The transplanted BMSCs migrated to the ischemic hemisphere along the corpus callosum and to the border of the infarction, which was confirmed by MRI and Prussian blue staining. The changes of infarction volume were not significantly different among these three groups. CONCLUSIONS: MRI is feasible for in vivo tracking of BMSCs labeled with SPIO in rats. The stem cell therapy may not be able to affect the volume of cerebral infarction.

[Gene transfer efficiencies and cell tropism of recombinant adeno-associated virus in the hippocampus of adult rat].

OBJECTIVE: To explore the gene transfer efficiencies and different cell tropism of recombinant adeno-associated virus 1 (rAAV1), rAAV 2, and rAAV 5 in the hippocampus of adult rats, and select more suitable gene vectors for central nervous system (CNS) gene therapy. METHODS: Eighteen SD male adult rat were divided into 3 groups randomly (n = 6), with every group being injected with the titre and volume matched rAAV1, rAAV2, and rAAV5 vectors. All these vectors contained enhanced green fluorescent protein (EGFP) sequences as a reporter gene. Animals were killed after 8 weeks. The coronal cryosections of brains were processed, and the EGFP gene expression was observed with fluorescence microscopy; the expression area and the number of EGFP positive cells were automatically measured using Image-Pro Plus 4.5 software. To identify their cell tropism in the CNS, the sections were counterstained with neuronal marker neuron-specific nuclear protein (NeuN) and the astrocyte marker glial fibrillary acidic protein (GFAP) and were examined with a confocal laser scanning microscope. RESULTS: Eight weeks after adeno-associated virus gene transfer, the expression profile of EGFP demonstrated significant difference. Most of the pyramidal cell layers of CA1 to CA3 area and granular cell of dent gyrus were strongly transduced by rAAV1; whereas rAAV2 primarily transduced the cells of multiform layer in hilar region of the dentate gyrus; only a few pyramidal cells were transduced by rAAV5. Moreover, rAAV1 showed significantly wider distribution throughout the hippocampus, and the quantity of EGFP positive cells and the EGFP positive area were significantly more than those of rAAV2 and rAAV5 (P < 0.01). The counterstaining for NeuN and GFAP showed that rAAV1 was able to transduce both neurons and glia cells, whereas rAAV2 and rAAV5 transduced the neurons only. CONCLUSION: rAAV1 is an excellent transgene vector with higher efficiency and broader cell tropism in the CNS.

[Distribution and expression of peroxisome proliferator activated receptor gamma in human pituitary adenomas].

OBJECTIVE: To evaluate the distribution and expression of peroxisome proliferator activated receptor gamma (PPAR-gamma) in human pituitary adenomas. METHODS: Thirty eight consecutive surgically resected pituitary adenomas and 5 normal pituitary tissues were enrolled in the study. Immunohistochemistry was used to confirm the distribution of PPAR-gamma. Expression of PPAR-gamma was evaluated by Western blot. RESULTS: PPAR-gamma immunoreactivity was located in the nucleoli of pituitary adenoma cells. PPAR-gamma was expressed in all human pituitary adenomas and normal pituitary tissues. Its expression in pituitary adenomas was significantly higher than in normal pituitary tissues (P < 0.01), and its expression in ACTH-secreting adenomas was significantly higher than in any other type of pituitary adenomas (P < 0.05). CONCLUSIONS: PPAR-gamma may play an important role in the generation, growth, and invasion of human pituitary adenomas. It may become a novel therapeutic target for these tumors.

[Factors influencing the recurrence of Cushing's disease treated by transsphenoidal surgery].

OBJECTIVE: To explore factors influencing the recurrence of patients with Cushing's disease after transsphenoidal surgery. METHODS: We retrospectively analyzed the clinical data of 182 patients treated by transsphenoidal surgery with Cushing's disease in our department in PUMC Hospital from 1992 to 2002. RESULTS: The recurrence rates were significantly different when patients had different radiological findings (P = 0.001), operative methods (P = 0.001), histological findings (P = 0.04), and postoperative cortisol levels (P = 0.02); however, such difference was not found in term of tumor size (P = 0.43). CONCLUSION: Radiological findings, operative methods, histological findings, and postoperative cortisol estimates may be the factors influencing the recurrence of patients treated by transsphenoidal surgery.

[Study on trans-differentiation of adult human myoblasts into neural precursor cells and its implantation in rats].

OBJECTIVE: To investigate the feasibility of inducing adult human myoblasts into neural precursor cells. METHODS: The myoblasts were isolated with mixed digestive enzyme from minced human temporal muscle samples, cultured and purified clonally. The 3rd passage cells were incubated with serum free medium including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and leukemia inhibitory factor (LIF). Morphological change was investigated during incubation period. Immunofluorescence cytochemistry and RT-PCR analysis were used to assess cell differentiation and trans differentiation. RESULTS: After the induction, cells became non-adherent aggregates as neurospheres. The myoblast-derived neurospheres was immuno-positive for nestin. In differentiation condition, they looked like neurons and glial cells and expressed neuronal (microtubule associated protein 2, MAP-2), astrocytic (Glial fibrillary acidic protein, GFAP) and oligodendrocytic (Galactocerebroside, Galc) markers by immunocytochemistry. The result by RT-PCR was coincident with immunocytochemistry. The myoblast-derived neurospheres expressed MAP-2 and GFAP after they were transplanted into the brain of rats with cerebral ischemia. CONCLUSION: Adult human myoblasts can be inducted to trans-differentiate into neural precursor cells.

[Expression of galectin-3 in invasive prolactinomas].

OBJECTIVE: To investigate the expression of galectin-3 (Gal-3) in prolactinomas. METHODS: Expressions of Gal-3 were evaluated by immunohistochemistry using polyclonal antibody in 16 invasive prolactinomas and 16 prolactinomas. RESULTS: Gal-3 was expressed both in invasive prolactinomas and noninvasive prolactinomas while significantly higher expression seen in the invasive prolactinomas (P < 0.05). CONCLUSION: Gal-3 expression may be used as a useful indicator to determine the invasiveness and prognosis of prolactinomas.

[Expression of enhanced green fluorescent protein in rat brain transduced by recombinant adeno-associated viruses type 1 and type 2].

OBJECTIVE: To explore the expression of enhanced green fluorescent protein (EGFP) transduced into the brain via recombinant adeno-associated virus (rAAV) type 1 and rAAV type 2 vectors so as to select the better rAAV serotype and feasible gene transfer route to central nervous system (CNS). METHODS: Twenty-four SD male adult rats were randomly divided into 4 equal groups: rAAV1 intra-hippocampus injection group, rAAV1 intra-ventricular injection group, rAAV2 intra-hippocampus injection group, and rAAV2 intra-ventricular injection group to be injected stereotactically with titer and volume matched rAAV1-EGFP and rAAV2-EGFP vectors respectively. The rats were sacrificed respectively 2 and 4 weeks after injection and their brains were removed to be made into serial frozen coronal sections. Fluorescence microscopy was used to observe the expression of EGFP in the brain and to calculate the expression volume of EGFP in different parts of the brain. RESULTS: Two weeks after injection EGFP was expressed in a small amount or not expressed in all groups. Four weeks after injection the EGFP expression volume were (7.00 +/- 0.98) mm(3) and (0.81 +/- 0.28) mm(3) in the rAAV1 and rAAV2 intra-hippocampus injection groups respectively (P < 0.01), and were (12.72 +/- 0.28) mm(3) and (0.24 +/- 0.13) mm(3) in the rAAV1 and rAAV2 intra-ventricular injection groups respectively (P < 0.001). CONCLUSION: As gene-transducing vector in CNS rAAV1 is superior to rAAV2. High expression can be achieved by intra-ventricular injection with rAAV1 vectors.

Elevated soluble epidermal growth factor receptor level in pituitary adenoma and carcinoma.

OBJECTIVE: To investigate effect of the soluble epidermal growth factor receptor (sEGFR/sErbB1) level in the peripheral blood in development, invasiveness, apoplexy of each type of pituitary tumor. METHODS: The sEGFR level was determined in peripheral serum from 190 patients with pituitary diseases by enzyme linked immunosorbent assay. The sEGFR levels were measured in 10 pituitary Rathke's pouch, 18 pituitary hyperplasia, 161 pituitary adenomas including 30 microadenomas, 83 large adenomas, 48 giant adenomas, 1 pituitary carcinoma, and 28 healthy controls. RESULTS: In the patients with pituitary hyperplasia, microadenoma, large adenoma, giant adenoma, and pituitary carcinoma, the sEGFR level was 188.92 +/- 32.62, 209.83 +/- 19.01, 333.20 +/- 69.33, 405.85 +/- 37.38, and 617.45 fmol/mL independently. They were all significantly higher than patients with pituitary Rathke's pouch (156.78 +/- 18.24 fmol/mL, P < 0.001) and healthy control group (159.11 +/- 40.50 fmol/mL, P < 0.05). The sEGFR level in pituitary carcinoma was higher than pituitary adenoma. In patients with pituitary adenoma, the sEGFR level was positive correlated to the size of pituitary adenomas (r=0.998), the significant difference was observed for the sEGFR level in each group of the patients with pituitary adenomas (P < 0.001). Furthermore, in patients with pituitary ACTH-secreting microadenomas, the serum sEGFR levels in invasiveness (295.00 +/- 77.80 fmol/mL) was higher than that in non-invasiveness (210.60 +/- 16.4 fmol/mL, P < 0.05). In patients with pituitary ACTH-secreting, PRL-secreting, GH-secreting, and non-functioning large adenomas, the serum sEGFR levels in invasiveness (407.86 +/- 28.50, 399.25 +/- 30.10, 386.00 +/- 13.08, and 369.25 +/- 36.70 fmol/mL) was higher than that in non-invasiveness (335.25 +/- 63.49, 300.64 +/- 47.57, 297.00 +/- 61.93, and 269.30 +/- 25.68 fmol/mL) respectively (P < 0.05). In patients with invasive pituitary PRL-secreting, GH-secreting, and non-functioning giant adenomas, the serum sEGFR levels not significantly different in between invasiveness (417.50 +/- 35.94, 409.50 +/- 69.14, and 417.50 +/- 44.13 fmol/mL) and non-invasiveness (386.00 +/- 49.64, 417.50 +/- 44.03, and 409.51 +/- 35.17 fmol/mL) (P > 0.05). In patients with pituitary large adenomas, the sEGFR levels in pituitary apoplexy (377.48 +/- 39.18 fmol/mL) was higher than that in non-pituitary apoplexy (343.18 +/- 68.17 fmol/mL, P > 0.05). CONCLUSIONS: The increased level of peripheral serum sEGFR is concomitant with development, proliferous size of the adenomas in patients with pituitary adenomas. In addition, the elevated levels of serum sEGFR occur in pituitary apoplexy as clinical active tumors, and the non-invasive ACTH secreting adenomas. The sEGFR levels could be differentiated helpfully between pituitary adenomas and non-pituitary adenomas. These data suggest that serum sEGFR could be as a referable marker of the size and activation of proliferation in pituitary adenoma.

[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.

[Expressive level of vascular endothelial growth factor in peripheral blood in patients with pituitary adenomas].

OBJECTIVE: To discuss whether vascular endothelial growth factor (VEGF) in the peripheral blood can reflect the biological activities of pituitary adenomas. METHODS: The concentrations of VEGF in peripheral blood were measured with ELISA in 203 patients with pituitary adenomas, 22 patients with pituitary hyperplasia, 7 patients with pituitary Rathke' pouch and 3 patients with pituitary abscess. RESULTS: The serum VEGF levels were (366.8 +/- 211.1) pg/ml and (286.8 +/- 107.6) pg/ml in patients with pituitary adenomas and pituitary hyperhasia, respectively, which were higher than those in patients with pituitary Rathke' pouch [(180.5 +/- 61.7) pg/ml], patients with pituitary abscess [(147.5 +/- 46.3) pg/ml] and the health control [(180.8 +/- 56.2) pg/ml] (P < 0.05). In patients with pituitary adenomas, the VEGF levels were (380.0 +/- 234.5) pg/ml in macroadenomas and (380.1 +/- 2870.3) pg/ml in giant adenomas, higher than those in microadenomas [(294.6 +/- 111.6) pg/ml] and in pituitary hyperhasia respectively (P < 0.05). The serum VEGF levels were not significantly different in pituitary adenoma in terms of invasive growth, apoplexy, cyst and hormone secretory functions (P > 0.05). CONCLUSIONS: The upregulation of serum VEGF expression may reflect the biological activities of pituitary adenoma. However, it may not be associated with pituitary Rathke' pouch, pituitary abscess, adenoma with invasiveness, apoplexy, cyst and hormone secretory function. The serum VEGF levels could be helpful in differentiating pituitary adenoma from pituitary Rathke' pouch and pituitary abscess.

[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.

[Measurement of epidermal growth factor receptor concentration in the pre- and postoperative serum in patients with meningiomas].

OBJECTIVE: To investigate effect of the serum concentration of epidermal growth factor receptor (EGFR) in the pre- and postoperative peripheral blood in patients with meningiomas. METHODS: Using ELISA, the EGFR concentration were measured in the pre- and postoperative serum in 53 patients with meningiomas. In this study, the patients were divided into preoperative group, postoperative group (including 42 total resection, 11 subtotal resection), and 28 healthy control. RESULTS: In 53 patients with meningiomas, concentration of preoperative serum EGFR was (352.93 +/- 66.18) fmol/ml, to show higher than control group (159.11 +/- 40.50) fmol/ml (P < 0.0001); Concentration of postoperative serum EGFR was (220.74 +/- 70.63) fmol/ml, to show lower than preoperative group (P < 0.001). Including in 42 patients with meningomas by total resection, serum EGFRs of the 38 patients were decreased (191.20 +/- 32.13) fmol/ml, the 4 patients with peritumoral edema were decreased (248.75 +/- 10.31) fmol/ml, to show lower than preoperative group (P < 0.001); the 11 patients with subtotal resection were decreased (322.14 +/- 89.53) fmol/ml, not to show different than preoperative group (P < 0.05). CONCLUSIONS: In 92.16% patients with meningiomas, the more poison of the tumor is resected, the less the serum EGFR concentration is detected postoperatively, while the total resection of mangionmas resulted in a lowest level of EGFR. These results support the concept that human meningiomas may be autocrine EGFRs. The measurement of serum EGFR can be useful to patients with meningiomas for follow-up after surgery.