A novel mutation in proprotein convertase subtilisin/kexin type 9 gene leads to familial hypercholesterolemia in a Chinese family

Background Familial hypercholesterolemia （FH） is an autosomal disorder associated with elevated plasma low density lipoprotein （LDL） levels leading to premature coronary heart disease （CHD）. As a result of long-term hyperlipemia, FH patients will present endarterium thickening and atherosclerosis. In the present study we scanned the related gene of a clinically diagnosed autosomal genetic hypercholesterolemia family for the possible mutations and established eukaryotic expression vector of mutation of proprotein convertase subtilisin/kexin type 9 （PCSK9） gene with gene recombination technique to investigate the contributions of the variation on low density lipoprotein receptor （LDL-R） metabolism and function alternation.Methods Mutation detection was conducted for LDL-R, apolipoprotein B100 （apoB100） and PCSK9 gene with nucleotide sequencing in a Chinese FH family. The full-length cDNA of wild type PCSK9 gene （WT-PCSK9） was obtained from Bel-7402. Site mutagenesis was used to establish the recombinant eukaryotic expression vector carrying pathogenic type of PCSK9 gene and the inserted fragment was sequenced. With the blank vector as control, liposome transfection method was used to transfect the Bel-7402 cells with recombinant plasmid. The expression of LDL-R mRNA was examined by RT-PCR. PCSK9 and the expression of LDL-R protein were determined by Western blotting. Results The G→T mutation at the 918 nucleotide of PCSK9 gene resulted in the substitution of the arginine by a serine at the codon 306 of exon 6. After sequencing, it was confirmed that the inserted fragment of established expression vector had correct size and sequence and the mutant was highly expressed in Bel-7402 cells. There was no significant variation in the levels of LDL-R mRNA. LDL-R mature protein was decreased by 57% after the cells were transfected by WT-PCSK9 plasmid. Mature LDL-R was significantly decreased by 12% after the cells were transfected by R306S mutant as evidenced by gray scale scanning, suggesting that the new mutant R306S can significantly decrease the expression of mature LDL-R protein.Conclusions A novel missense mutation of PCSK9 gene, R306S, was found and the eukaryotic expression vectors of mutant and wild-type of PCSK9 gene were established. There was no significant variation in the levels of LDL-R mRNA. The R306S mutation could significantly lead to the decrease of LDL-R mature protein expression, which might be the pathogenic gene of the FH family.     

Analysis of low-density lipoprotein receptor gene mutations in a Chinese patient with clinically homozygous familial hypercholesterolemia

Objective To screen the point mutation of the low-density lipoprotein receptor （LDL-R） gene in Chinese familial hypercholesterolemia (FH) patients, characterize the relationship between the genotype and the phenotype and discuss the molecular pathological mechanism of FH. Methods A patient with clinical phenotype of homozygous FH and her parents were investigated for mutations in the promoter and all eighteen exons of the LDL-R gene. Screening was carried out using Touch-down PCR and direct DNA sequencing; multiple alignment analysis by DNASIS 2.5 was used to find base alteration, and the LDL-R gene mutation database was searched to identify the alteration. In addition, the apolipoprotein B gene (apo B) was screened for known mutations (R3500Q) that cause familial defective apo B100 (FDB) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).Results Two new heterozygous mutations in exons 4 and 9 of the LDL-R gene were identified in the proband (C122Y and T383I) as well as her parents. Both of the mutations have not been published in the LDL-R gene mutation database. No mutation of apo B100 (R3500Q) was observed. Conclusion Two new mutations (C112Y and T383I) were found in the LDL-R gene, which may result in FH and may be particularly pathogenetic genotypes in Chinese people.     

Detection of ATP2C1 Gene Mutation in Familial Benign Chronic Pemphigus

Summary： The ATP2C1 gene mutation in one ease of familial benign chronic pemphigus was investigated.One patient was diagnosed as familial benign chronic pemphigus by pathology, ultrastructral examination and clinical features. Genomic DNA was extracted from blood samples. Mutation of ATP2CI gene was detected by polymerase chain reaction （PCR） and DNA sequencing. The results showed that deletion mutation was detected in ATP2C1 gene in this patient, which was 2374delTTTG. No mutation was found in the family members and normal individuals. It was coneluded that the 2374delTTTG mutation in ATP2C1 gene was the specific mutation for the clinical phenotype for this patient and was a de novo mutation.     

INTRAVASCULAR ULTRASOUND IMAGING OF ANGIOGRAPHICALLY "SILENT" LEFT MAIN CORONARY ARTERY ATHEROSCLEROSIS

Patients with left main coronary artery (LMCA) atherosclerosis have a poor prognosis compared with lesions in the other coronaries. Because of the methodological problems. LMCA atherosclerotic lesions are not frequently detected by coronary angiography. The purpose of the study was to reveal the existence of LMCA disease in patients with normal coronary arteries by using intravascular ultrasound imaging. Ninety-seven patients with angiographically normal coronary arteries were examined with a 3.5 F or 4.8 F. 20 MHz intravascular ultrasound catheter. The vessel, lumen and plaque areas were determined and percent area and diameter stenosis were calculated. Plaque formation with or without calcific deposits identified by ultrasound accoustic shadowing were regarded as signs of atherosclerosis.     

Features of a Chinese family with cerebral cavernous malformation induced by a novel CCM1 gene mutation

Background Familial cerebral cavernous malformations （CCMs）, characterized by hemorrhagic stroke, recurrent headache and epilepsy, are congenital vascular anomalies of the central nervous system. Familial CCMs is an autosomal dominant inherited disorder and three CCM genes have been identified. We report a Chinese family with CCMs and intend to explore clinical, pathological, magnetic resonance imaging （MRI） features and pathogenic gene mutation of this family. Methods Totally 25 family members underwent brain MRI examination and clinical check. Two patients with surgical indications had surgical treatment and the specimens were subjected to histopathological and microstructural examination. In addition, polymerase chain reaction （PCR） and direct sequencing were performed with genomic DNA extracted from 25 family members＇ blood samples for mutation detection. Results Brain MRI identified abnormal results in seven family members. All of them had multiple intracranial lesions and four cases had skin cavernous hemangioma. T2-weighted sequence showed that the lesions were typically characterized by an area of mixed signal intensity. Gradient-echo （GRE） sequence was more sensitive to find micro- cavernous hemangiomas. There was a wide range in the clinical manifestations as well as the age of onset in the family. The youngest patient was an 8-year-old boy with least intracranial lesions. Histopathological and microstructural examination showed that CCMs were typically discrete multi-sublobes of berry-like lesions, with hemorrhage in various stages of illness evolution. They were formed by abnormally enlarged sinusoids and the thin basement membranes. A novel T deletion mutation in exon 14 of CCM1 gene was identified by mutation detection in the seven patients. But unaffected members and healthy controls did not carry this mutation. Conclusions The clinical manifestations were heterogenic within this family. We identified a novel mutation （c.1396delT） was the disease-causing mutation for this family and extended the mutational spectrum of CCMs.     

散发性甲状旁腺癌一例分子遗传学分析

Objective To analyze the clinical and molecular genetic characteristics of one patient with sporadic parathyroid carcinoma(s-PC).Methods The clinical profile,laboratory data and paraffinembedded tissue sample of a s-PC patient were collected at our hospital.Genomic DNA was extracted from the leukocytes of peripheral blood and paraffin-embedded tissue of this patient.All 17 exons of HRPT2 gene including the flanking regions of introns were amplified by PCR.The mutations of HRPT2 gene were analyzed by directly sequencing the amplified DNA fragments.Parafibromin encoded by HRPT2 gene was analyzed by immunohistochemistry.Results The patient was diagnosed as s-PC by the clinical presentations,laboratory examinations and typical pathologic characteristics.HRPT2 germline mutation was identified as a base mutation at codon 222(CGA＞TGA)and caused a nonsense mutation at the codon (R222X) resulting in a truncated protein.Parafibromin was completely lost while comparing the normal parathyroid tissues by immunohistochemistry.Condusion The altered expression of parafibromin caused by HRPT2 gene mutation is one of the molecular mechanisms for explaining the clinical manifestations of this patient.     

The Relationship of Insulin—receptor Gene Polymorphism to Ischemic Stroke？

Objective To investigate the role of mutation of insulin-receptor(INSR) gene in the development of ischemic stroke.Methods The base-variations at exon 17 and 20 of INSR gene,by means of PCR-SSCP were determined in 68 cases of atherothrombotic cerebral infarction (ACI),81 cases of lacunar infarction(LI) and 62 healthy controls(HC).Results There were 2 alleles of T and C at exon 17 of INSR gene.The prevalence of mutant of t allele in ACI patients was more common than that in the controls.the blood pressure and the parameters of lipid metabolism in the patients with mutant were higher than those in the controls with wild-type gene.However,the correlative analysis showed that the polymorphism of INSR gene was not related statistically to the blood pressure.No base-variation at exon 20 was found in the study.Conclusion The mutation at exon 17 of INSR gene,by promoting the development of atherosclerosis,may participate in the occurrence of ischemic stroke.     

散发性甲状旁腺癌一例分子遗传学分析

Objective To analyze the clinical and molecular genetic characteristics of one patient with sporadic parathyroid carcinoma(s-PC).Methods The clinical profile,laboratory data and paraffinembedded tissue sample of a s-PC patient were collected at our hospital.Genomic DNA was extracted from the leukocytes of peripheral blood and paraffin-embedded tissue of this patient.All 17 exons of HRPT2 gene including the flanking regions of introns were amplified by PCR.The mutations of HRPT2 gene were analyzed by directly sequencing the amplified DNA fragments.Parafibromin encoded by HRPT2 gene was analyzed by immunohistochemistry.Results The patient was diagnosed as s-PC by the clinical presentations,laboratory examinations and typical pathologic characteristics.HRPT2 germline mutation was identified as a base mutation at codon 222(CGA＞TGA)and caused a nonsense mutation at the codon (R222X) resulting in a truncated protein.Parafibromin was completely lost while comparing the normal parathyroid tissues by immunohistochemistry.Condusion The altered expression of parafibromin caused by HRPT2 gene mutation is one of the molecular mechanisms for explaining the clinical manifestations of this patient.     

New mutation detection system of repackaged λ gt11 DNA containing LacZ gene

Objective: To establish a reformative detection system which has sound ability of providing information on molecular mutagenesis spectrum and the specificity of detection system of repackaged λ phage. Methods: LacZ gene, as mutational target gene and reporter gene, was applied into the detection system. The λ gt11 DNA treated with ENU (1-ethyl-1-nitrosourea) and 9-AA (9-aminoacridine) was repackaged in vitro. The packaged λ phage was then grown in E. coli Y1090 on a selective plate containing X-gel and IPTG. The survival and mutation frequencies were determined by counting the clear-plaque and blue-plaque, and the molecular mutation mechanism was studied by extracting and sequencing the LacZ gene of mutants. Results: The survival of repackaged λ phages treated with 9-AA and ENU apparently decreased in consistent dose-dependence. The mutation frequency of clear-plaque mutants showed a linear dose-related increase. The predominant mutations induced by 9-AA were ± 1 frameshift mutation, and 9-AA induc     

Association of Polymorphisms in Angiotensin-converting Enzyme and Type 1 Angiotensin Ⅱ Receptor Genes with Coronary Heart Disease and the Severity of Coronary Artery Stenosis

To explore the relation of angiotensin-converting enzyme （ACE） and angiotensin Ⅱ type 1 receptor （AT1R） gene polymorphism with coronary heart disease （CHD） and the severity of coronary artery stenosis, 130 CHD patients who underwent coronary angiography were examined for the number of affected coronary vessels （≥75% stenosis） and coronary Jeopardy score. The insertion/deletion of ACE gene polymorphism and AT1R gene polymorphism （an A→C transversion at nucleotide position 1166） were detected by using polymerase chain reaction （PCR） and restriction fragment length polymorphism （RFLP） in CHD patients and 90 healthy serving as controls. The resuits showed that DD genotype and of ACE were more frequent in CHD patients than that in control group （38.5% vs 14.4%, P〈0.001）. The frequency of the ATIR A/C genotypes did not differ between the patients and the controls （10% vs 13.1%, P〉0.05）. The relative risk associated with the ACE-DD was increased by AT1R-AC genotype. Neither the number of affected coronary vessels nor the coronary score differed among the ACE I/D genotypes （P〉0.05）. But the number of affected coronary vessels and the coronary score were significantly greater in the patients with the AT1R-AC genotype than in those with the AA genotype （P〈0.05）. In conclusion, DD genotype may be risk factor for CHD and MI in Chinese people, and is not responsible for the development of the coronary artery stenosis. The AT1R-C allele may increase the relative risk associated with the ACE-DD genotype, and may be involved in the development of the stenosis of coronary artery.     

Relationship between coronary artery remodeling and cumulative incidence of coronary angiographic lesions with vulnerable characteristics in patients with stable angina pectoris

ackground Development of vulnerable lesions is not limited to the target lesions, but a pan-coronary process. Such lesions are identified by positive remodeling （intravascular ultrasound （IVUS） and complex lesions （angiography））. The prevalence of lesions with vulnerable characteristics in patients with stable angina was not well known. The purpose of the present study was to evaluate the relationship between coronary artery remodeling and incidence of angiographic complex lesions and its calcification in stable angina patients.Methods One hundred and sixty-one stable angina patients （95 males, aged （68±11） years） with 161 de novo target lesions were studied using pre-interventional IVUS. Remodeling index was defined as the lesion divided by reference vessel area; positive remodeling was defined as remodeling index 〉1.05. Besides the 161 target lesions, there were 613 angiographic lesions with 〉30% diameter stenoses, classified as complex or smooth. Multiple complexes were defined as more than one complex lesion in one patient. Stenoses of at least 70% were described as tight. Calcium arc area was used as a new method to quantify coronary calcification.Results Fifty-six patients had positive remodeling target lesion, while 105 did not. The overall number of lesions with a diameter stenoses 〉30% was similar in patients with or without positive remodeling, and the frequency of angiographically complex lesions was higher in positive remodeling patients, especially at non-target site. Calcium arc area was smaller in patients with positive remodeling.Conclusions Positive remodeling on intravascular ultrasound was associated with more complex lesions angiographic findings, especially at non target site. Positive remodeling was found less calcified in patients with stable angina.     

Comparative study on 16-slice CT coronary angiography vs conventional coronary angiography—A report of 38 cases

The clinical application of 16-slice CT coronary angiography （CTCA） and the impact of plaques differently characterized on assessing coronary artery stenosis were evaluated. Thirty-eight patients with coronary artery disease diagnosed by conventional coronary angiography （CAG） underwent 16-slice CTCA （collimation： 16×0.75 mm; rotation time： 420 msec; kernel： 35f; effective current： 500 mAs; tube voltage： 120 kV）. The interval between CTCA and CAG was within one month. CTCA was evaluated by consensus of two independent experienced radiologists unknowing CAG findings. Original images, maximum intensity projections and multiplanar reconstructions were used to assess coronary artery stenosis. For a determined plaque an attenuation value ≥ 130 HU was considered as calcified, and 〈130 HU noncalcified. The plaques were then classified into significant calcification （extensive calcification）, medium calcification （small isolated calcification） and noncalcification. The diagnostic accuracy of 16-slice CTCA findings as well as to detect ≥50% stenoses caused by plaques was evaluated respectively regarding CAG as the standard of reference. In comparison with CAG findings, the sensitivity, specificity, positive and negative predictive value derived from CTCA for mild stenosis （〈50%） were 72.7%, 38.5%, 50%, 62.5%, respectively; for moderate stenosis （50%-75%） 82.4%, 72.7%, 70%, 84.2%, resepctively; and for severe coronary stenosis （〉75%） 85%, 90.5%, 81%, 92.7% respectively. With the increase of stenoses degree, the value of CTCA was greater. For the classification of the plaque calcification with ≥50% stenosis CTCA attained the sensitivity, specificity, positive and negative predictive value for severe calcificatoin 73.3% 22.2%, 61.1% and 33.3%, respectively; for moderate calcification 70%, 55.6%, 63.6% and 62,5%, respectively; for noncalcification 93.8%, 85.7%, 93.8% and 85.7% respectively. CTCA was restricted in assessing coronary artery stenosis in the presence of calci     

Analysis of the inhibitory effect of gypenoside on Na+, K+-ATPase in rats’ heart and brain and its kinetics

Objective： To study the effects of gypenoside （Gyp） on the activity of microsomalNa^＋, K^＋-ATPase in rat＇s heart and brain in vitro. Methods： The microsomal Na^＋, K^＋-ATPase was prepared from rat＇s heart and brain by differential centrifugation. The activity of microsomal Na^＋, K^＋-ATPase was assayed by colorimetric technique. Enzyme kinetic analysis method was used to analyze the effect of Gyp on the microsomal Na^＋, K^＋-ATPase of rats. Results： Gyp reversibly inhibited the brain and heart＇s microsomal Na^＋, K^＋-ATPase in a concentration-dependent manner, and showed a more potent effect on enzyme in the brain. The IC50 of Gyp for the heart and brain were 58.79± 8.05 mg/L and 52.07± 6.25 mg/L, respectively. The inhibition was enhanced by lowering the Na^＋, or K^＋-concentrations or increasing the ATP concentration. Enzyme kinetic studies indicated that the inhibitory effect of Gyp on the enzyme is like that of competitive antagonist of Na^＋, the counter-competitive inhibitor for the substrate ATP, and the mixed-type inhibitor for K^＋. Cenclusien： Gyp displays its cardiotonic and central inhibitory effects by way of inhibiting heart and brain＇s microsomal Na^＋, K^＋-ATPase activities in rats.     

Gene Transfection Mediated by Ultrasound and Pluronic P85 in HepG2 Cells

In order to assess whether gene transfection could be mediated by ultrasound in associa- tion with P85 and find the appropriate parameters of ultrasound irradiation, the effects of ultrasound with or without P85 on gene transfection of HepG2 cells were examined. The HepG2 cells were irra- diated by ultrasound at 1 MHz, 0.4-2.0 W/cm2 and 50% duty cycle with plasmid encoding enhanced green fluorescent protein (EGFP) as a report gene. Forty-eight h later, the expression of EGFP was detected under the fluorescence microscopy. Transfection efficacy was quantitatively assessed by flow cytometry, and cell viability was evaluated by trypan blue exclusion. The results showed that the transfection efficacy was increased with the increases in ultrasound output power and the ideal trans- fection efficacy was achieved in HepG2 cells irradiated by ultrasound at 0.8 W/cm2 for 30 s. The transfection efficacy in ulstrasound P85 group was three times higher than in single ultrasound group [(17.63±1.07)% vs (5.57±0.56)%, P<0.05]. The cell viability was about 81% and 62% in ultrasound group and ultrasound P85 group respectively. It was concluded that ultrasound in combination with P85 could mediate the gene transfection of HepG2 cells, ideal transfection efficacy was achieved by ultrasound irradiation at 0.8 W/cm2 for 30 s, and P85 could somewhat increase the damage to cells caused by ultrasound.     

The clinical application of multi-slice spiral CT angiography in abdominal aortic disease

Objective： To evaluate the clinical application of multi-slice spiral CT angiography（MSCTA） in the assessment of abdominal aortic disease. Methods： Fifty-four patients underwent multi-slice spiral CT angiography of abdomen. Contrast agent （Omnipaque 300 I g/L） 1.5 ml/kg was injected and the injection rate was 3 ml/s. The delay time was determined by bolus tracking technique, Tll level abdominal aorta was set as the target vessel and the threshold was 180-200Hu, slice width was 3mm and with a pitch of 4-6. Original data were transferred to working-station to perform functional reconstruction. Results： Ten cases were normal, twenty-eight cases were abdominal aortic aneurysms, five abdominal aortic dissecting aneurysms （Debakay type Ⅲ） and eleven aortic sclerosis. SSD showed the body of aneurysm and the relationship between aneurysm and adjacent blood vessel, MIP better displayed calcification of blood vessel wall and condition of the stent, MPR demonstrated true and false lumen, rapture site of abdominal aorta intima and mural thrombus. Conelusion： MSCTA axial and reconstruction image can show the extent of abdominal aortic disease and the relationship with adjacent blood vessels. It is a safe, simple and non-invasive examination method.     

MOLECULAR ANALYSIS OF RADIATION-INDUCED MUTATION IN EXON 7/8 OF RAT HPRT GENE

Objective To investigate the relationship between the radiation dose and the HPRT gene lo-cus mutation in rat smooth muscle cells, and provide the molecular basis for prevention of restenosis after percutaneous transluminal coronary angioplasty (PTC4). Methods The smooth muscle cells cultured in vitro were irradiated by radionuclide 188Re in different doses. HPRT gene mutation colonies were selected and isolatedby 6-thioguanine. Analysis of mutation in exon 7/8 of HPRT gene were accomplished by polymerase chain reaction and single-strand conformation polymorphism. Results The HPRT gene mutation frequency of rat smooth muscle cells that were irradiated by radionuclide 188Re ranged from 5.5× 10-6 to 13 ×10-6. Of 91 HPRT gene mutation colonies, 13 (14.3%) contained exon 7/8 deletion and 15(16.5%) had point mutation.The exon 7/8 mutation frequency was 30.8% . There were significant relationships between radiation dose and mutation frequency of HPRT gene and exon 7/8 . Conclusion The DNA damage and gene mu     

Sequence analysis on drug-resistant rpoB gene of Mycobacterium tuberculosis L-form of isolated from pneumoconlosls workers

To study the characteristics of drug-resistant genetic mutation of rpoB on coal workers＇ pneumoconiosis complicated with L-form of Mycobacterium tuberculosis. Methods： A total of 42 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected, including 31 drug-resistant strains. Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator. Results： No mutation of rpoB gene was identified in 11 rifampicin-sensitive strains while conformation changes were found in 31 rifampicin-resistant strains. The mutation rate was 93.55% （29/31） in resistant strains, mainly concentrated in codon 531 （51.6%, 16/31） and 526 （32.26%, 10/31）. Base substitutions happened, including 27 unit point mutation and 2 two point mutation. The mutation of codon 516 that new found wasn＇t reported by internal and overseas scholars. Conclusion： The substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for rifampicin resistance in Mycobacterium tuberculosis L-forms. It also proves that rpoB gene is diversiform.     

Twelve years’ experience and clinical results of using the radial artery for coronary revascularization

Background The radial artery （RA） is becoming a popular conduit for coronary artery bypass grafting （CABG）,yet data reporting the long-term results are rare.We reported our clinical,angiographic and intravascular ultrasound findings on 93 patients who had the RA used as part of the conduit for the CABG procedures during a 12-year period from June 2001 to June 2013.Methods A total of 118 radial artery conduits were harvested in 87 males and 6 females,age from 28 to 66 （mean 49.9） years.An ＂intra-operative Allen＇s test＂ was developed to safeguard blood supply to the arm and hand.A ＂double-clip ＆ scissors-cut＂ technique was carried out to minimize the thermal injury to the radial artery from the diathermy.The left radial artery was used in 67 patients,the right in one,and bilateral radial arteries in 25 patients.One hundred and twenty-two out of 272 distal anastomoses （44.9％） were constructed with radial arteries,with an average of 2.9 grafts per patient （range 2-6）.Results Follow-up angiography and intravascular ultrasound study at 3-139 postoperative months （mean 59 months） revealed a 93.1％ RA patency.String sign occurred in one patient in whom the RA was directed to a big right coronary artery with a stenosis of around 50％.The patency for the internal mammary artery was 96.4％.Conclusions The RA is an excellent conduit that broadens the options for total arterial CABG surgery.Good graft patency could be achieved through careful harvesting techniques and choice of proper target coronary vessels.     

The Protective Effects of In Vitro Cultivated Calculus Bovis on the Cerebral and Myocardial Cells in Hypoxic Mice

The protective effects of in vitro cultivated calculus bovis （ICCB） on the cerebral and myocardial cells in hypoxic mice and the mechanism were examined. In one group, mice were intragastrically （i.g.） given ICCB for 15 days and then they were subjected to acute cerebral ischemia by decapitation, and then the panting time was recorded. In the other group, 12 min after exposure to hypoxia, mice was administered the ICCB i.g. for 5 days, and then the blood serum and tissues of brain, heart, liver were harvested and examined for SOD, GSH-px and T-AOC activity and content of MDA. The tissues of brain and heart were observed electron-microscopically for ultrastructural changes. The corpus striatum and hippocampus of brain were collected and examined for content of dopamine （DA） and norepinephrine （NE）. The ultrastrural examination showed that the pathological change in brain and heart in the ICCB group was very slight, while abnormal changes in the control group were obviously more serious. ICCB significantly prolonged the panting time of the hypoxic mice （P〈0.001）, increased the activity of SOD, GSH-px, T-AOC in serum and tissues of brain, liver, heart and elevated the content of DA and NE. ICCB also pronouncedly reduced content of MDA in serum and tissues of brain, heart and liver. Significant differences in these parameters were noted between ICCB group and controls. It is concluded that ICCB can exert protective effect on the cells of brain and myocardium by enhancing the tolerance of the tissues to hypoxia and the body＇s ability to remove free radicals and regulating the neurotransmitters.     

Ultrasound-mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization

Background Many studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization （CNV）, and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor （PEDF） could inhibit CNV.
Methods Human retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDFgene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure （intravitreal injection）; infusing liposomes with the naked plasmid DNA of PEDF into the vitreous （lipofectamine ＋ PEDF）; infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately （retrobular injection）; infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately （vein injection）. The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography.
Results In vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with microbubbles or ultrasound alone. In the rat CNV model, transfection efficiency mediated by ultrasound/microbubbles was significantly higher than that by lipofectamine-mediated gene transfer at 28 days after treatment. The study also showed that with the administration of ultrasound-mediated microbubbles destruction, the CNV of rats was inhibited effectively.
Conclusions Ultrasound-microbubble technique could increase PEDF gene transfer into rats＇ retina and chorioid, in association with a significant inhibition of the development of CNV, suggesting that this noninvasive gene transfer method may provide a useful tool for clinical gene therapy.