Human ß-NGF gene transferred to cat corneal endothelial cells.

AIM: To transfect the cat corneal endothelial cells (CECs) with recombinant human ß-nerve growth factor gene adeno-associated virus (AAV-ß-NGF) and to observe the effect of the expressed ß-NGF protein on the proliferation activity of cat CECs. METHODS: The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco's modified Eagle's medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-ß-NGF was constructed. The recombinant human AAV-ß-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-ß-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the ß-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope. RESULTS: The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human ß-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-ß-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-ß-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result showed that the percentage of G1cells in CEC-AAV-NGF group was 76.8% while that in normal CEC control group and CEC-AAV control group was 46.6% and 49.8%. CONCLUSION: Recombinant AAV-ß-NGF promotes proliferation in cat CECs by expressing bioactive ß-NGF protein in high efficiency and suggests that its modulation can be used to treat vision loss secondary to corneal endothelial dysfunction.

Effect of recombinant human platelet-derived growth factor B on cat corneal endothelial cell viability mediated by adeno-associated virus.

AIM: To transduce recombinant human platelet-derived growth factor B(PDGF-B) gene adeno-associated virus(AAV) to in vitro cultured cat corneal endothelial cell (CEC) and observe the effect of the expressed PDGF-BB protein on the viability of cat CEC. METHODS: Cat cornea endothelium was torn under microscope and rapidly cultivated in DMEM to form single layer CEC and the passage 2 endothelial cells were used in this study. The recombinant human PDGF-B gene AAV was constructed and transduced into cat CEC directly. Three groups were as following: blank control group, AAV control group and recombinant AAV group. At 24 hours, 48 hours, and 5 days after transduction, total RNA was extracted from the CEC by Trizol and the expression of PDGF-B gene was detected by fluorescence quantitative polymerase chain reaction. Viability of the transduced CEC was detected at 48 hours after transduction by MTT assay. Cell morphology was observed under inverted phase contrast microscope. RESULTS: With the torn endothelium culture technique, we rapidly got single layer cat CEC. At 24 hours, 48 hours and 5 days after transduction, fluorescence quantitative polymerase chain reaction showed there was no significant difference of the expressed PDGF-B gene mRNA between blank control group and AAV control group (P>0.05). In contrast, there were significant differences between two control groups and recombinant AAV group (P<0.05). MTT assay showed that in recombinant AAV group, the expressed PDGF-BB protein could promote the viability of cat CEC. Morphology observation showed at 48 hours after transduction, cells in CEC-AAV-PDGF-B group proliferated into bigger scales in regular triangle to hexagon shape with distinct boundary, while the number of cells was significantly less in the two control groups. CONCLUSION: The recombinant AAV-PDGF-B expresses biological active PDGF-BB protein in cat CECs, which promotes the viability and proliferation of cells.

The study of human PDGF-B gene transferred to cat corneal endothelial cells.

AIM: To demonstrate that human platelet-derived growth factor-B (PDGF-B) cDNA could be expressed in primary cultured cat corneal endothelia cells by using gene transfer techniques; to explore a useful tool for the further studies of the molecular mechanisms of corneal endothelium failure and provide a potential effective genetic therapy for the blind patients. METHODS: Human PDGF-B cDNA was isolated from human placent by RT-PCR and inserted into pcDNA(4) vector to construct recombinant eukaryotic expression plasmid pcDNA(4)-PDGF-B. The full length was confirmed by the DNA sequencing analysis. By tearing endothelium technique we obtained pure single layer of cat corneal endothelial cells. The pcDNA(4)-PDGF-B eukaryotic expression vector was transferred into cat corneal endothelial cells by Effectene™ lipofectine. The transfection efficiency of Effectene™ lipofectine in pcDNA(4)-B was detected with pcDNA(4)-GFP. 5 days later, RT-PCR was used to check the PDGF-B expression. Cell viability was tested by modified tertrozalium salt (MTT) method. Cell morphology was observed under inverted phase contrast microscope. RESULTS: The human PDGF-B cDNA was isolated successfully from healthy parturien placent tissue and the sequence was confirmed by computer automatic sequence and PCR analysis. Pure single layer cat corneal endothelial cells were successfully cultured by tearing endothelium technique. Effectene™ lipofectine transfection technique could be effectively used to transfer pcDNA(4)-PDGF-B into cat corneal endothelial cells in vitro, the transfection efficiency was 30%. RT-PCR result showed that human PDGF-B gene was highly expressed in transfected cat corneal endothelial cells. The expressed PDGF-BB protein promoted the viability of cat corneal endothelial cells. CONCLUSION: Human platelet-derived growth factor-B (PDGF-B) cDNA could be highly expressed in cultured cat corneal endothelial cells by gene transfection techniques. Expressed PDGF-BB protein significantly promoted the viability of cat corneal endothelial cells, thus provided a potential effective method for corneal endothelium blindness genetic therapy.

Effect of basic fibroblast growth factor on cat corneal endothelial cell proliferation.

AIM: To investigate the function of basic fibroblast growth factor (bFGF) on cat corneal endothelial cells proliferation. METHODS: Cat corneal endothelial cells were primarily cultured, stimulated with bFGF for different period, the proliferation of cells was assayed by modified tertrozalium salt (MTT) method, and the morphologic changes were observed with inverted phase contrast microscope and transmission electron microscope. RESULTS: At 1, 3 and 5 days after bFGF was added to cat corneal endothelial cells, the result of MTT in 490nm showed significant difference than that in control group, and the difference was most significant in 10ng/mL group. CONCLUSION: bFGF can promote proliferation of cat corneal endothelial cells. 10ng/mL is the relatively most effective dose.

Cloning, expression and functional analyses of human platelet-derived growth factor-B chain peptide for wound repair of cat corneal endothelial cells.

OBJECTIVE: To investigate the biological function of platelet-derived growth factor B (PDGF-B) on the survival and proliferation of cat corneal endothelial cells so as to provide bases for further studies of its role in wound repair and its clinical application. METHODS: Total RNA was extracted from the placenta tissues of healthy pregnant women undergoing hysterotokotomy and PDGF cDNA was obtained with reverse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-PDGF-B was constructed and expressed the recombinant PDGF-B in Escherichia coli (E. coli) BL21 (DE3). After purification and refolding on Ni2+-chelation affinity chromatography (NTA) column, it was used to culture cat corneal endothelial cells. Cell proliferation was tested by modified tertrazolium salt (MTT) and flow cytometer. And the morphologic change and the ultrastructure were observed under an inverted phase contrast microscope, a scanning electron microscope and a transmission electon microscope, respectively. RESULTS: PDGF-B chain peptide (PDGF-BB) gene was successfully inserted into the prokaryotic expression vector, pET-28a (+). The purified recombined protein pET-PDGF-B showed a single band on sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the deduced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal endothelial cells. CONCLUSIONS: The construction of recombinant prokaryotic expression vector pET-PDGF-B and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB protein. The expressed PDGF-BB promotes the proliferation of cultured cat corneal endothelial cells.

Efficacy and safety of benidipine therapy of essential hypertension in elderly Chinese patients.

BACKGROUND: Benidipine (CAS 105979-17-7) is a dihydropyridine calcium channel blocker used in the treatment of hypertension and angina pectoris. OBJECTIVE: To examine the efficacy and safety of therapy with benidipine in elderly hypertensive patients. METHODS: Chinese patients >60 years of age with mild to moderate essential hypertension were enrolled. The patients were prescribed benidipine at the dose of 8 mg once daily for 12 weeks. Detailed laboratory examinations and 24-h ambulatory blood pressure monitoring were performed before and after the treatment. RESULTS: One hundred and sixty-four of the 180 patients enrolled completed the 12-week active treatment phase. Sitting systolic blood pressure (SBP) and diastolic blood pressure (DBP) reductions at the end of treatment were 21.50 +/- 12.83 and 10.60 +/- 8.04 mmHg, respectively; the proportion of patients showing a good treatment response was 95.1% for SBP and 96.9% for DBP. Benidipine significantly reduced the mean 24-h ambulatory blood pressure (p < 0.001 vs. baseline) exhibiting smooth, sustained effects and high trough-to-peak ratios (T/P ratio) (0.87 for SBP and 0.72 for DBP). Moreover, benidIpine significantly reduced the systolic morning blood pressure surge and urinary albumin, and it was well tolerated. No serious adverse events were noted. CONCLUSION: Benidipine was welltolerated and effective in elderly Chinese patients with essential hypertension.

[Proliferation and regeneration effects of constructed human beta-nerve growth factor gene on transfected cat corneal endothelial cells in vitro].

OBJECTIVE: To explore the mechanisms of proliferation and regeneration effects of a human nerve growth factor (beta-NGF) expression vector (pcDNA4-beta-NGF) on the transfected cat corneal endothelial cells in vitro. To provide a new method for long term cultivation of human corneal endothelial cells in vitro and to establish theoretical basis of gene therapy for corneal endothelial defects. METHODS: It was a experimental study. The human pcDNA4-beta-NGF expression vector was constructed and transfected into cultured cat corneal endothelial cells by Effectene lipofectine transfection technique. The expression of the reporter gene pcDNA4-beta-LacZ expression was used to determine the transfection efficiency 48 hours after the transfection. RT-PCR and immunohistochemistry techniques were used to check the transient expression status at mRNA and protein levels in cat corneal endothelial cells. Mitotic index and methyl thiazolyl tetrazolium (MTT) value were measured and cell numbers at different stages of cell cycles were determined by flow cytometer 96 hours after transfection. An in vitro quantitative cat corneal endothelial cell traumatic model was established which was used for observing the effect of human beta-NGF expression product on the DNA synthesis of cat endothelial cells and healing process of traumatized endothelial cells. RESULTS: A human nerve growth factor (beta-NGF) expression vector (pcDNA4-beta-NGF)was successfully constructed and confirmed by sequence analysis. Single layered pure cat corneal endothelial cells were obtained by a modified sliced tissue culture technique and confirmed by morphological analysis, neurone specific enolase immunohistochemistry study and transmission electronic microscope. Effectene lipofectine mediated transfection efficiency of pcDNA4-beta-NGF into cat corneal endothelial cells in vitro was 11.3%. The human beta-NGF could be highly expressed in the transfected corneal endothelial cells at mRNA and protein levels. Mitotic index, MTT value and G1 stage cell numbers, as well as traumatically defected endothelial cells numbers during the healing process of human beta-NGF transfected corneal endothelial cells were statistically differed from the pre-transfected cells and control groups. CONCLUSIONS: Effectene lipofectine transfection technique could be effectively used for transfecting pcDNA4-beta-NGF into cat corneal endothelial cells in vitro with good efficacy and the gene could stably express to improve the proliferation and regeneration of the cat corneal endothelial cells. This method could be managed as an experimental basis to be applied in the experimental study for transfecting the human beta-NGF gene into human corneal endothelial cells. Therefore a new method for resolving the problem of impossible regeneration of corneal endothelial cells could become possible.

[Inhibition of corneal neovascularization by vascular endothelial growth inhibitor gene].

OBJECTIVE: To evaluate the effect of Effectene lipofectene mediated plasmids encoding human pcDNA4-vascular endothelia growth inhibitor (pcDNA4-VEGI) gene on corneal neovascularization (CNV). METHODS: It was an experimental study. Forty healthy New Zealand albino rabbits (40 eyes) were divided into 4 teams according to the random digits table: team A (10 rabbits) was the team which transfected by lipofectine mediated plasmids encoding human pcDNA4-vascular endothelia growth inhibitor (pcDNA4-VEGI) gene; team B (10 rabbits) was the team transfected by empty vector; team C (10 rabbits) was the team transfected by lipofectine; team D (10 rabbits) was the empty control group which was transfected by saline. (1) Human VEGI gene fragment was connected with expressional vector plasmid pcDNA4 to construct the pcDNA4-VEGI gene expression vector. Computer automatic sequence analysis was used to identify the gene. (2) New Zealand albino rabbits were sutured by 5 - 0 silk in the superior cornea to establish the animal model and were transfected by pcDNA4-VEGI gene mediated by Effectene lipofectene transfection. Length and area of CNV were measured by slit lamp every day after transfection, immunohistochemistry was used to detected the expression of VEGI protein in cornea at the time 3, 7, 14 and 21 d. RESULTS: (1) Computer automatic sequence analysis confirmed the correct recombination of pcDNA4-VEGI gene. (2) Average occurrence of CNV in the pcDNA4-VEGI gene transfected team was 6.3 days while that in the control teams were 3.1, 3.2, 3.2 days (F = 39.838, P < 0.01). Average length and area of CNV were also different in the VEGI team and the control teams (q between team A and B on average length of CNV was 17.386, 20.944 and 8.892 on 7, 14 and 21 d; q between team A and C on average length of CNV was 19.488, 19.795 and 7.483 on 7, 14 and 21 d; q between team A and C on average length of CNV was 19.583, 20.413 and 8.941 on 7, 14 and 21 d, and P were all < 0.01. And q between team A and B on average area of CNV was 30.238, 57.820 and 35.543 on 7, 14 and 21 d; q between team A and C on average area of CNV was 32.607, 57.843 and 36.653 on 7, 14 and 21 d; q between team A and C on average area of CNV was 33.873, 57.590 and 34.724 on 7, 14 and 21 d, and P were all < 0.01). Integrated epithelium, light stroma edema, less CNV and less inflammation could be seen in the gene-therapy team. And immunohistochemistry results showed VEGI positive cells in epithelium, stroma, endothelium and the cliff of CNV in the gene therapy team. CONCLUSIONS: PCR, enzyme cutting, recombination and other genetic techniques could be used to construct expressional pcDNA4-VEGI gene. Effectene lipofectene transfection technique could be effectively used in transfecting pcDNA4-VEGI gene into rabbit cornea to inhibit CNV.

[Expression of apoptosis related genes and changes of lacrimal functions in main lacrimal gland extirpation xerophthalmia model].

OBJECTIVE: To establish a main lacrimal gland extirpation xerophthalmia model and to study the relationship between expression of apoptosis genes in various ocular surface cells (cornea and conjunctiva epithelium cells and Meibomian cells) and ocular surface sicca as well as tissue damages in this model. METHODS: Twenty adult Wistar rats were included in this study and allocated to two groups randomly. Main lacrimal glands were excised in 10 animals and Schirmer I test, tear film break-up time (BUT) and fluorescence staining were performed before the operation and 3 days, 1, 2, 4, 8 and 12 weeks after the operation. Ten experimental animals and ten normal animals in the control group were investigated by immunohistochemistry staining of the cornea, conjunctivas epithelium cells and Meibomian cells to detect the expression of Bax and Bcl-2 gene. RESULTS: Schirmer I test and BUT were significantly lower in the experimental group 1-2 weeks after the operation. The difference became more prominent in the course of observation (P < 0.05). Fluorescence staining was positive in the experimental group. Cornea, conjunctival epithelial cells and Meibomian cells showed more increased expression of Bax in the experimental group [(503.47 +/- 343.73), (586.36 +/- 296.47), (436.61 +/- 246.96) every thousands cells] than that in the normal controls [(241.71 +/- 130.12), (311.03 +/- 142.33), (202.16 +/- 109.29) every thousands cells] (P < 0.05). Bcl-2 expression was decreased in the experimental group [(237.32 +/- 87.07), (236.07 +/- 81.24), (251.79 +/- 89.93) every thousands cells] as compared to the normal controls [(474.24 +/- 167.62), (342.81 +/- 114.57), (320.42 +/- 153.32) every thousands cells] (P < 0.05). CONCLUSIONS: The apoptosis of the epithelial cells in cornea, conjunctiva and Meibomian cells may be one of the mechanisms lead to the destruction of ocular surface tissues in xerophthalmia.

[Molecular cloning and expression of human PDGF-B chain peptide gene and its effect on cat corneal endothelial cell proliferation in vitro].

OBJECTIVE: To construct pET-PDGF-B plasmid with over expression of PDGF and investigate its effects on cat corneal endothelial cells proliferation in vitro. METHODS: Gene rearrangement technique was used to construct the prokaryotic vector with insertion of pET-PDGF-B. The recombinant PDGF-B was obtained from E. coli BL21 (DE3). The effects of recombinant PDGF-B on proliferation and morphologic changes of cultured cat corneal endothelial cells were assayed by modified tertrozalium salt (MTT), inverted phase contrast microscope and transmission electron microscope. RESULTS: PDGF-B peptide gene was inserted into prokaryotic expression vector pET-28a (+), which was confirmed by sequence analysis; a 27,000 protein band was demonstrated as pET-PDGF-B from extraction of E. coli BL21 (DE3) using western blot; MTT assay showed PDGF-BB promoted the proliferation of cat corneal endothelial cells. CONCLUSIONS: A constructed pET-PDGF-B plasmid vector over expression of PDGF is produced. Recombinant PDGF-BB promotes cultured cat corneal endothelial cells proliferation.

Animal study on expression of laminin and fibronectin in cornea during wound healing following alkali burn.

OBJECTIVE: To observe the expression of laminin and fibronectin in alkali-burned corneas in rats. METHODS: A total of 18 normal Wistar rats were randomly divided into 6 groups (n = 3 in each group). For each rat, one eye was injured by alkali burn, the other one was taken as the normal control. Then all the corneas were surgically removed and the expression of laminin and fibronectin was observed with immunohistochemistry respectively at 7 hours, 1 day, 3 days, 7 days, 14 days and 28 days after alkali burn. RESULTS: Compared with that of the normal controls, the expression of laminin and fibronectin of the burned eyes was dramatically higher at 7 hours, reached peak at 14 days and decreased to the normal level at 28 days after alkali burn. CONCLUSIONS: In the process of wound healing after alkali burn, the expression of laminin and fibronectin increases dramatically, which suggests that laminin and fibronectin may participate in the process of corneal wound healing.