Insect antifreeze protein (AFP) has high antifreeze activity. Antifreeze proteins can be used in cryopreservation of biological tissues and cells. We expressed an antifreeze protein from the desert beetle Microdera punctipennis in yeast and determined the function of the protein at low temperatures. Yeast expression vector, pPIC9K-Mpafp698, was constructed and transformed into Pichia pastoris GS115. The expression of MpAFP698 was induced by methanol, and identified by tricine SDS-PAGE and Western blotting. Mpafp698 gene was inserted into the genome of the host yeast strain GS115, and correctly expressed. Hardly any yeast's own protein was secreted into the media. Cryoprotective experiments showed that MpAFP698 can significantly protect mouse liver as well as other mouse organs from cold damage compared with those in the control of Bovine serum albumin (BSA) addition. Besides, the hemolysis of blood cells protected by MpAFP698 at 4 degrees C was reduced and the survival rate of SF9 cells protected by MpAFP698 after freezing and thawing was increased compared to those of the control with BSA addition. Our results showed that MpAFP698 can be expressed in yeast, which allows a convenient purification of the MpAFP protein that has the cryoprotective effect.
Animals ; Antifreeze Proteins ; biosynthesis ; Blotting, Western ; Cold Temperature ; Coleoptera ; Cryoprotective Agents ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Freezing ; Insect Proteins ; biosynthesis ; Mice ; Pichia ; metabolism ; Sf9 Cells

Objective To evaluate the role of contrast-enhanced color Doppler ultrasonography with Levovist in the diagnosis of liver tumors.Methods One hundred and twenty-five patients with 126 hepatic solid lesions were examined by conventional color Doppler and contrast enhanced ultrasonography.All hepatic lesions were confirmed by pathology.Of these,79 cases with hepatocellular carcinoma,4 cases with metastatic liver carcinoma,13 cases with hemangioma,7 cases with inflammatory pseudotumor of liver,7 cases with focal nodule hyperplasia,and 15 cases with other benign lesions.The Levovist with a concentration of 400 mg/ml was injected into an antecubital vein within approximately 2 minutes.Results All hepatic solid lesions with blood supply were differently enhanced by contrast-ultrasonography (I～IV grades).The color flow signals with grade III and grade IV were detected in one malignant lesion before contrast-enhancement and in 41 malignant lesions ( 49.4%) after contrast-enhancement.The color flow signals were detected by contrast-enhanced sonography in 26 malignant lesions ( 92.8%) with no color Doppler signals and in 33 benign lesions ( 76.7%).The mean resistive index of liver cancer group was much higher than that of benign lesion group before and after contrast-enhancement.The accuracy rate for diagnosing liver tumors was raised from 66.7% (84/126) in pre-enhancement to 94.4% (119/126) in post-enhancement.Conclusions The contrast-enhanced color Doppler ultrasonography is very useful in the differential diagnosis of liver tumors.

Objective To use the antisense of Platelet-derived growth factor ? chain (PDGF-?) gene to inhibit the proliferation of rabbit vascular smooth muscle cells(VSMCs) in order to provide in situ basis on prevention and treatment of human restenosis. Methods A rabbit restenotic vascular model was constrcted and an antisense designed according to PDGF-? cDNA sequence as a drug was used to observe its effects on the expression of proliferating cell nuclear antigen and in situ expression of platele-derived growth factor ? chain by the VSMCs and the formation of neointima after injury. Results The antisense could significantly inhibit the expression of proliferating cell nuclear antigen and downregulate in situ expression of PDGF-? mRNA by intimal VSMCs one week after injury with inhibitory rates of 93.44% and 88.40%, respectively. Conclusion The antisense designed could inhibit the formation of neointima after injury through downregulating the expression of PDGF-? mRNA by local VSMCs, which provides the experimental basis of the antisense for the prevention and treatment the human restenosis.

AIM: To elucidate the co-transfection of platelet derived growth factor B(PDGF-B) antisense oligonucleotide and tissue-type plasminogen activator gene to prevent vascular anastomotic restenosis after coronary bypass.METHODS: A dog model of vascular anastomotic restenosis after coronary bypass was constructed. A constructed tissue-type plasminogen activator(tPA) gene plasmid and a designed PDGF-B oligonucleotide were used to transfect into the dog cardiomyocytes and anastomotic vascular smooth muscle cells(VSMCs) at the same time of coronary bypass, using a therapeutic ultrasound for the gene delivery. Effects of these two genes on thrombosis in local anastomotic vessels, the expressions of proliferating cell nuclear antigen(PCNA) and PDGF-B mRNA by VSMCs and the proliferation of vascular intima were observed with the methods of routine pathological, immuno-histochemical staining, in situ hybridization and morphometry. RESULTS: PDGF-B antisense oligonucleotide and tissue-type plasminogen activator gene were succesfully transfected. These two genes significantly inhibited the expressions of PCNA and PDGF-B mRNA in intimal VSMCs with the inhibitory rates of 65.01% and 81.75%, respectively. The local intimal thickness and area also reduce markablely and the thrombosis of the anastomosis was prevented followed by the reduction of the anastomotic restenotic rate of 62.63%. CONCLUSION: Co-transfection of PDGF-B antisense oligonucleotide and tissue-type plasminogen activator gene inhibits the dog experimental anastomotic restenosis after coronary bypass.

Objective To evaluate the feasibility of virtual touch tissue quantification (VTQ) for the assessment of renal allograft.Methods A total of 72 kidney recipients were examined with conventional ultrasound and VTQ after transpantation.Biopsies were performed in 34 patients,20 patients were with acute rejection (AR),14 with chronic allograft nephropathy (CAN),38 patients as control group.The peak systolic velocity (PSV) and resistance index(RI) were measured on main,infrarenal and arcuate arteries with conventional ultrasound and shear wave velocity (SWV) of the renal cortex was obtained by VTQ.All the data were compared among three groups.Results There were no significant differences of PSV between two groups.An increased RI was presented in the CAN group(P ＜0.05).The mean SWV was (2.67 ± 0.27) m/s,(2.90 ± 0.31)m/s and (2.28 ± 0.24)m/s for AR,CAN and normal group,respectively.There were significant differences of SWV among the three groups (P ＜ 0.05).Conclusions VTQ technique could provide a new method for the assessment of transplanted kidney.

Objective To probe the correlation between the short-axis diameter(SD) of coagulation necosis and the microwave power(P) using a new type of internally cooled microwave antenna in ex vivo and in vivo liver model.Methods Fourteen microwave ablations were performed in swine livers ex vivo and canine livers in vivo under the microwave powers of 30-90W and the durations of 10 min and 20 min, respectively.The short-axis diameter(SD) and the long-axis diameter(LD) of the coagulation were measured.The difference of SD, LD and SD/LD in swine liver ex vivo and canine liver in vivo were compared.The relationship between the SD and microwave power was analysised using linear regression model.Results Under the same conditions,the SDs and LDs of the coagulations of canine livers in vivo were smaller than those of swine livers in ex vivo (P <0.01).There was such a higher linear relationship(r = 0.96-0.99, P <0.01) between the SD of coagulation and the power in vivo liver model as the results acquired in ex vivo.Conclusions Although the blood flow decreased the SDs of the coagulation of microwave ablation in liver in vivo, but there was still a higher linear relationship between SD and microwave power.

Objective To investigate the efficacy of endoscopic intranasal structure reconstruction surgery for rhinogenous headache. Methods 82 cases of rhinogenous headache were given endoscopic intranasal structure reconstruction. Compared VAS scores before and 6 months after treatment, counted the effective rate. Results Among the 82 patients, 69 patients(84. 1%) were cured, 8 cases (9. 8%) were of obvious effect, 5 cases (6. 1%) were invalid, and the total efficiency was 93. 9%. VAS scores of the mucosal contact headache group before treatment were greater than that of the sinus headache group (P<0. 05). Compared with VAS scores before treatment, both of the two groups had lower VAS scores after treatment (P<0. 05), but there was no significant difference between the two groups after treat-ment (P>0. 05). Conclusion Endoscopic surgical operation can remove the extrusion of the nasal cavity and paranasal sinuses and factors of nasal congestion, and then reconstruct normal anatomical structure of nasal cavity, thus restoring normal function of paranasal sinuses. It has good therapeutic effect on rhinogenic headache.

Objective To probe the correlation between the short-axis diameter(SD) of microwave coagulation and the time(T) and/or power(P) using a new type of internally cooled microwave antenna. Methods A MTC-3 microwave apparatus with a new type of internally-cooled microwave antenna was used to coagulate a fresh porcine liver in vitro. The correlation was analyzed between the SD of coagulation necrosis and the time and/or power. Results ①Under the same power, there was high linear relationship(r= 0.94- 0.98,P

AIM: To elucidate the in vivo mechanisms of the proliferation of vascular smooth muscle cells (VSMCS) in injuried arteries. METHODS: A VSMCS proliferative model was constructed by injury of rabbit iliac arteries with balloon catheters and a probe designed for rabbit platelet-derived growth factor B chain (PDGF-B ) mRNA was used to detect the expression of it by intimal VSMCS on the vascular cross sections using an in situ hybridization technique at the indicated times. The relation of this expression to the proliferation of VSMCS by their expression of proliferating cell nuclear antigen (PCNA) and vascular intimal areas were estimated. RESULTS: The expression of PDGF-B mRNA of intimal VSMCS was increased when calculating the intimal PDGF-B mRNA positive cells per millimetre area at ?400 magnification with average numbers of 31.93?14.64 in 1 week group, 26 50?9 25 in 2 weeks group and 24 85?13 65 in 4 weeks group. This was in accordance with the expression of PCNA by VSMCS and the increase of intimal areas. CONCLUSION: The local production of PDGF-B by VSMCS via an autocrine mechanism is responsible for the continuous proliferation of these cells and formation of neointima after the injury. The probe designed is very useful for detecting rabbit PDGF-B mRNA.

0.05 ), those in the malignant group were 53%(18/34) and 94%(32/34) with a distinguished improvement after enhancement(P