Land use function change and its driving force of the "production-living-ecological" space in Fenhe River Basin from 1980 to 2020.

It is of importance to analyze land use function change and driving factors of the production-living-ecological space of national territory to realize the coordinated development. Based on land use remote sensing data in 1980, 1990, 2000, 2010, and 2020, we used the methods of land use dynamics, transfer matrix, center of gravity shift and geographic detector to analyze the pattern of production-living-ecological space in Fenhe River Basin and explore the influencing factors. The results showed that the area proportion of production-living-ecological space in the basin was ecological space > production space > living space from 1980 to 2020. The ecological space and agricultural production land showed a decreasing trend, with a decrease of 72441.19 and 105882.96 hm2, respectively. The living space and industrial production land showed an upward trend, with an increase of 119503.02 and 58821.13 hm2, respectively. There was significant difference in the land use function change of production-living-ecological space. Agricultural production land had the largest transferred area, accounting for 47.9% of the total. The largest transfer area of industrial land was agricultural land, which accounted for 61.3% of the total from 2000 to 2010. The occupation of agricultural land by urban living land was mainly distributed in the marginal area of various urban areas of Taiyuan Basin. Among them, the increasing area of urban living land in Taiyuan City showed a trend of gradual southward expansion. The center of gravity migration of urban land for living and industrial production land was the most obvious, and that for living showed the trend of first moving south and then moving north, while industrial production land moved northward significantly. The influence of social-economic factors on the land use change was obviously stronger than that of natural factors, while the interaction between social-economic factors had a stronger explanatory power. The results would provide reference for clarifying the relationship between land function transformation and optimizing land use function of production-living-ecological space.

[Spatial-temporal differentiation of mountain-water-forest-farmland-lake-grass system in Qinghai area of the Qilian Mountain National Park, China]. / ç¥è¿žå±±å›½å®¶å ¬å›­é’æµ·ç‰‡åŒºå±±æ°´æž—ç”°æ¹–è‰çš„æ—¶ç©ºåˆ†å¼‚.

Higher and more precise requirements are critically needed for the protection, regulation, and restoration of ecological environment in the Qilian Mountain National Park after it is classified as a national park system pilot in China. Based on remote sensing data in 1980-2018, the spatial pattern map of mountain-water-forest-farmland-lake-grass system was constructed to analyze its spatial-temporal variations in the general control area and core conservation area in Qinghai area of the Qilian Mountain National Park. The results showed that grasslands, with an area of 8174.93 km2, were the main landscape in the park, and that grassland area in the core conservation area was 1.2 times as that of the general control area. The bare exposed rocks, a major type of unused land, accounted for 86.7% and 79.4% of the unused land in the core conservation area and the general control area, respectively. Forest area in the general control area was larger than that in the core conservation area. Water area in the core conservation area was 4.9 times as large as that in the general control area, with 90.4% of which being dominated by permanent glaciers and snowfields. The drylands were mainly concentrated in the general control area. From 1980 to 2018, the water area was decreasing and had been reduced by 186.75 km2. The area of permanent glaciers and snowfields decreased the most, with a drop of 12.05 and 175.88 km2 in the general control area and the core conservation area, respectively. The area of forests and grasslands were enlarged constantly. The changes of high-, medium-, and low-coverage grasslands in the core conservation area were greater than that in the general control area, which were the most significant during 1990-2000. Moreover, the degradation of high- and medium-coverage grasslands in the general control area as well as high- and low-coverage grasslands in the core conservation area was observed from 1980 to 2018. The area of bare exposed rocks was on the rise, while the permanent glaciers and snowfields displayed a decreasing trend. The permanent glaciers and snowfields and the bare exposed rocks exhibited the most obvious changes in the park. The glaciers in the core conservation area retreated remarkably faster than those in the general control area, which were transformed into the bare exposed rocks mainly in 1980-1990 and 2000-2010.

[Changes of growing season NDVI at different elevations, slopes, slope aspects and its relationship with meteorological factors in the southern slope of the Qilian Mountains, China from 1998 to 2017]. / 1998­2017年祁连山南坡不同海拔、坡度和坡向生长季NDVIå˜åŒ–åŠå ¶ä¸Žæ°”è±¡å› å­çš„å ³ç³».

Qilian Mountains is an important water conservation area in Northwest China, which is the boundary between the first and second steps of China's topography and is sensitive to climate change. Based on the data of temperature, precipitation, normal difference vegetation index (NDVI), and digital elevation model (DEM) data, we analyzed NDVI change and its relationship with temperature and precipitation along the elevation, slope and slope aspect in the southern slope of Qilian Mountains using tendency analysis method, wavelet analysis and correlation analysis. The results showed that, from 1998 to 2017, NDVI value of the growing season presented increasing trend by a rate of 0.023·10 a-1. Changes of NDVI differed at different elevations, slopes and slope aspects. NDVI increased first and then decreased with elevation. The vegetation coverage at 2700-3700 m was good, and degraded in the area of >4700 m. NDVI reduced with the increases of slope, which showed little difference in different slope aspects but was better in sunny slope than in shade slope. NDVI of the growing season was closely related with temperature and precipitation. NDVI, temperature and precipitation in growing season all had a 14-year cycle. Vegetation at different elevations, slopes and slope aspects was differently affected by temperature and precipitation. Vegetation in areas with altitude <3700 m, >4700 m, slope <25° and each slope direction was more sensitive to precipitation.

[Land use change and its driving force on the southern slope of Qilian Mountains from 1980 to 2018]. / 1980­2018å¹´ç¥è¿žå±±å—å¡åœŸåœ°åˆ©ç”¨å˜åŒ–åŠå ¶é©±åŠ¨åŠ›.

Qilian Mountains is the boundary of the first and second steps of China's terrain, with fragile ecological environment. There is great ecological significance to study land use change and driving force in transitional areas. In this study, we investigated the spatial and temporal characte-ristic of land use and its driving force in the south slope of Qilian Mountains, based on RS image data from 1980 to 2018, with the spatial autocorrelation method, ArcGIS spatial analysis method and principal component analysis. The results showed that, from 1980 to 2018, grassland was the main land use type, and the proportion of construction land was the smallest. Water area and grassland showed a declining trend, while unused land, construction land and farmland showed an increasing trend. There was smaller change for the woodland. The single dynamic degree of different land types decreased following an order of construction land, water, farmland, unused land, woodland and grassland. The comprehensive dynamic degree of land use was 0.9%. The spatial distribution of different land use types showed the characteristics of spatial agglomeration. The increased areas of farmland and the decreased areas of woodland and grassland were mainly distributed in the northwest of the Datong River valley of Menyuan County, while in the upper reaches of Datong River in the northeast of Tianjun County, grassland was occupied by construction land. The driving force of land use was population, science and technology, urbanization, level of economic development, and policies. Our results would support the government to reasonably plan and utilize land resources, which is of significance to the protection of ecological environment and the sustainable development of society and economy on the southern slope of Qilian Mountains.

ClCRY2 facilitates floral transition in Chrysanthemum lavandulifolium by affecting the transcription of circadian clock-related genes under short-day photoperiods.

Plants sense photoperiod signals to confirm the optimal flowering time. Previous studies have shown that Cryptochrome2 (CRY2) functions to promote floral transition in the long-day plant (LDP) Arabidopsis; however, the function and molecular mechanism by which CRY2 regulates floral transition in short-day plants (SDPs) is still unclear. In this study, we identified a CRY2 homologous gene, ClCRY2, from Chrysanthemum lavandulifolium, a typical SDP. The morphological changes in the C. lavandulifolium shoot apex and ClFTs expression analysis under SD conditions showed that adult C. lavandulifolium completed the developmental transition from vegetative growth to reproductive growth after eight SDs. Meanwhile, ClCRY2 mRNA exhibited an increasing trend from 0 to 8 d of SD treatment. ClCRY2 overexpression in wild-type (WT) Arabidopsis and C. lavandulifolium resulted in early flowering. The transcript levels of the CONSTANS-like (COL) genes ClCOL1, ClCOL4, and ClCOL5, and FLOWERING LOCUS T (FT) homologous gene ClFT1 were upregulated in ClCRY2 overexpression (ClCRY2-OE) C. lavandulifolium under SD conditions. The transcript levels of some circadian clock-related genes, including PSEUDO-REPONSE REGULATOR 5 (PRR5), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1), and GIGANTEA (GI-1 and GI-2), were upregulated in ClCRY2-OE C. lavandulifolium, while the expression levels of other circadian clock-related genes, such as EARLY FLOWERING 3 (ELF3), ELF4, LATE ELONGATED HYPOCOTYL (LHY), PRR73, and REVEILLE8 (RVE8), were downregulated in ClCRY2-OE C. lavandulifolium under SD conditions. Taken together, the results suggest that ClCRY2 promotes floral transition by fine-tuning the expression of circadian clock-related gene, ClCOLs and ClFT1 in C. lavandulifolium under SD conditions.

Role of CD80 in stimulating T lymphocyte activation.

AIM: To observe biological characteristics of hepatocarcinoma cells before and after CD80 transfection and to compare the effect of CD80-transfected hepatocarcinoma cells on T lymphocyte activation. METHODS: Retro virus vector carrying CD80 gene was transfected into HepG2 cells to establish CD80-transfected hepatocarcinoma cells (HepG2/hCD80). Flow cytometry (FCM) was performed to detect CD80 expression in the transfected cells. RT-PCR was used to evaluate CD80 expression at mRNA level. In the presence of anti-CD3 mAb, the proliferation of T lymphocyte was observed by MTT. Meanwhile, the expression of activated molecule marker CD25 was analyzed through FCM. RESULTS: A stable cell line HepG2/hCD80 expressing the human CD80 was established. Growth curve showed that the molecule CD80 could obviously decrease the growth of tumor cells. HepG2/hCD80 was evidenced to have a potency to enhance T cell proliferation and upregulate CD25 expression. CONCLUSION: CD80 transfection can lower malignant phenotype of hepatocarcinoma cells. CD80 transfection has a down-regulatory effect to activated T cells in vitro.

[Quantitative analysis of gene expression for vascular endothelial growth factor and its application].

Vascular endothelial growth factor (VEGF), a central mediator of angiogenesis, not only plays an important role in the pathogenesis of leukemia, but also is an independent prognostic factor in patients with hematologic malignancies, like those in solid tumors. However, the importance of VEGF during differentiation or apoptosis of leukemia cells remains to be elucidated. In order to assess the alternation of VEGF gene expression in the process of all-trans retinoic acid (ATRA)-induced differentiation of NB4 acute promyelocytic leukemia cell line, and a competitor DNA fragment, VEGF gene competative template (T-VEGFDelta) was constructed by using gene recombinant technologies, and a competitive quantitative reverse transcriptase-polymerase chain reaction (cQRT-PCR) method was developed. A standard curve was obtained by co-amplification of serial dilutions of the target nulecules with constant amount of competitive template and this curve was used to detect molecular number of target gene in measuring sample. The surface expression of CD11b antigen and nitroblue tetrazolium (NBT) reduction rate of NB4 cells were also assayed at different time points. The results showed that cQRT-PCR was a sensitive, reliable tool for analysis of VEGF gene expression with a detectable range from 1 x 10(4) to 2 x 10(5) molecules. The number of VEGF gene transcripts detected by means of cQRT-PCR assay was 42.3 x 10(5), 12.6 x 10(5), 3.6 x 10(5), and less than 1.0 x 10(5)/microg total RNA at 0, 12, 24 and 48 hours after ATRA treatment, respectively. This rapid down-regulation of VEGF gene expression, during ATRA-induced NB4 cell differentiation, was accompanied by the up-regulation of CD11b expression and an increased NBT reduction rate. In conclusion, cQRT-PCR method was successtully constructed, confirming that ATRA efficiently repressed VEGF, at the same time, the ATRA might exert an antileukemic effect, other than induction of differentiation via inhibition of angiogenesis.

[The expression of CXCR4 on acute leukemia cells and its implication for extramedullary infiltration].

OBJECTIVE: To study the expression of CXCR4 in acute leukemic cells and its clinical significance. METHOD: Bone marrow samples from 73 acute leukemia patients and leukemic cell lines were investigated by flow cytometry (FCM), the expression of SDF-1 in human marrow stromal cells and meninges were studied by using reverse transcription polymerase chain reaction (RT-PCR). Adhesion, migration and invasion of U937, NB4 and K562 cells were studied in vitro. RESULTS: The expression rates of CXCR4 in ALL and AML patients was 65.6% and 17.1%, respectively. And it was 0.2%, 41.0% and 52.0% in K562, U937 and NB4 cells, respectively. The extramedullary infiltration rates were 61.9% and 18.2% for CXCR4 positive and negative groups of ALL, respectively (P < 0.05); while in AML, the number of peripheral white blood cells in CXCR4 positive group was lower than that in CXCR4 negative group (P < 0.05). SDF-1alpha could enhance the adhesion, migration and invasion capacity of leukemic cells in vitro. CONCLUSION: Overexpression of CXCR4 in AL cells might be the molecular mechanism of extramedullary infiltration in leukemia.

[Establishment and characterization of leukemic cell line U937 stably expressing exogenous RbAp46].

To establish leukemic cell lines stably transfected by RbAp46 gene, electroporation was performed after optimizing the transfection condition for suspended cells. Under conditions of low voltage and high capacitance, RbAp46 was transfected into U937 by electroporation. Individual clones selected with G418 for 3 weeks were isolated. The integration and the protein levels of the exogenous RbAp46 in transfectants were determined by PCR and Western blot analysis, respectively. The subclone expressing high level of RbAp46 was then established. Viability of transfected cells was assayed by trypan blue exclusion. Cell number was counted daily to determine the growth rate. The results showed that growth rate of U937 cell lines expressing exogenous RbAp46 was about 50% lower than that in control. It is concluded that leukemic cell lines stably expressing exogenous RbAp46 were established and overexpression of RbAp46 inhibits the growth of U937 leukemic cells.

Expression of survivin and its significance in colorectal cancer.

AIM: To study the expression of survivin, a novel member of inhibitors of apoptosis protein (IAP) and its significance in colorectal carcinoma. METHODS: Survivin mRNA expression was evaluated by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in 52 colorectal carcinoma samples and 48 adjacent normal colorectal tissue samples. PCR product was sequenced to verify the desired result. Expressions of survivin protein, proliferating cell nuclear antigen labelling index (PI) and apoptotic index (AI) were detected immunohistochemically in 52 human colorectal carcinomas. RESULTS: The expression of survivin mRNA was detected in a significantly greater proportion of colorectal carcinoma samples than in adjacent normal colorectal tissues (67.3% vs 25%; P<0.01). There was no relationship between survivin mRNA expression in colorectal carcinomas and sex, tumor size, histological types, lymphnode metastasis, distant metastasis and Dukes' stage. The PCR product shared 99% of homology with human counterparts. Survivin expression was observed immunohistochemically in 27 of 52 cases of colorectal carcinoma (51.9%). The AI was significantly lower in survivin positive group than in survivin negative group (0.67+/-0.18% vs 1.14+/-0.42%; P<0.001), while the PI was greater in survivin positive group than in survivin negative group (51+/-22% vs 27+/-18%, P<0.001). CONCLUSION: Survivin is a special tumor marker independent of histopathological characteristics. It may play an important role during human colorectal tumorigenesis by inhibiting apoptosis and accelerating proliferative activity of colorectal tumor cells.

[Report of a case of congenital plasminogen activator inhibitor-1 deficiency].

OBJECTIVE: To report a patient with congenital plasminogen activator inhibitor-1 (PAI-1) deficiency and explore its molecular mechanism. METHODS: The activities of tissue plasminogen activator (tPA), alpha(2) antiplasmin (alpha(2)AP) and PAI-1 were measured by the methods of chromogenic substrate, the antigens of tPA and PAI-1 were measured by ELISA. PAI-1 gene was studied by PCR product sequencing and restriction endonuclease ana-lysing. RESULTS: In the present patient, the euglobulin clot lysis time was 70 minutes and was corrected to normal range after added 50 ng/ml PAI-1 to his plasma. The activities of t-PA, alpha(2)AP, and factor were normal; the activity and antigen of PAI-1 in plasma were both significantly decreased. Nucleotide sequence analysis revealed that the patient had a heterozygous missense mutation in exon 2, a G to A transition at nucleotide 43. The possibility of gene polymorphism was excluded by restriction endonuclease analysing. CONCLUSIONS: It is the first patient with congenital PAI-1 deficiency reported in China. The PAI-1 deficiency in the patient may be caused by compound heterozygosity, one of which is the G to A transition at nt43, a new mutation in congenital PAI-1 deficiency.

[Effects of tributyrin on SHI-1 leukemia cells in vitro].

OBJECTIVE: To investigate the effects of tributyrin (TB), a histone deacetylase inhibitor, on the growth, differentiation and apoptosis of SHI-1 leukemia cells and explore its possible mechanism. METHOD: Cell proliferation and viability were determined by cell counting, trypan blue dye exclusion. Cell morphological analysis, Annexin binding, DNA electrophoresis, expression of CD11b and CD14, NBT reduction were performed to evaluate differentiation and apoptosis of SHI-1 cells. The level of acetylated histone H3 was detected by Western blot and p21(WAF1/CIP1) expression by semi-quantitative RT-PCR. RESULTS: TB inhibited the proliferation and viability of SHI-1 cells in a time-dose dependent manner. The morphology of SHI-1 cells cultured in the presence of 0.1 mmol/L TB for 72 hs was more mature with higher NBT positivity and up-regulated expressions of CD11b and CD14 than that of control group. Exposed to 0.5 - 1.0 mmol/L TB for 48 hs, SHI-1 cells exhibited the morphological hallmarks of apoptosis, the increasing of Annexin binding and the DNA ladder on gel electrophoresis. The level of acetylated histone H3 and p21(WAF1) mRNA extracted from SHI-1 cells were increased by the treatment of TB. CONCLUSION: TB can inhibit proliferation, induce differentiation and apoptosis of SHI-1 cells. The mechanism may associate with its up-regulation of acetylated histone and the expression of p21(WAF1).

[Combined effects of endostatin gene transfer and ionizing radiation on lung adenocarcinoma model of A549-cells].

OBJECTIVE: The combined inhibition effects of endostatin gene transfer and ionizing radiation on lung adenocarcinoma model of A549-cell were investigated. METHODS: Human endostatin gene was transferred into lung adenocarcinoma A549 cell by retrovirus-mediation to obtain an A549/Endo cell. A549 and A549/Endo cells were xenoimplanted in nude mice respectively (each group included 10 mice). Then, the changes of the tumor size of each group (n = 5) and its growth inhibition rate were estimated. The implanted tumors of each group (n = 5) were exposed to radiation with 20 Gy at 28 day after implantation. They were irradiated again with 20 Gy 3 day later. The ionizing radiation was strictly confined to the tumor by shielding the rest of the body with lead. The size of tumor was measured periodically. The microvessel density (MVD) of 4 groups of implanted tumors (A549, A549/Endo, A549 + IR and A549/Endo + IR) were compared on day 42 postgrafting by the immunohistochemical method. RESULTS: PCR confirmed that endostatin gene was inserted into the genomic DNA of human lung adenocarcinoma A549 cell. The tumor formation time showed significant difference (P < 0.05) between group A549 (7.8 +/- 1.6) d and group A549/Endo (12.2 +/- 1.7) d. At the time of day 42 postgrafting, the tumor sizes of group A549 and group A549/Endo were (927.8 +/- 269.2) mm3 and (217.5 +/- 81.5) mm3 respectively (P < 0.01), and the tumor growth inhibition rate was 76. 5%. At the time of day 14 after irradiation, the tumor sizes of group A549 and group A549/Endo were (157.7 +/- 49.0) mm3 and (4.6 +/- 2.9) mm3 respectively (P < 0.01). The results of immunohistochemical detection showed that the MVD of-A549/Endo implanted tumor was significantly decreased [21.62 +/- 3.55 compared with A549 implanted tumor 35.78 +/- 5.67 (P < 0.01)]. Moreover , it also showed that ionizing radiation could further reduce the MVD of A549/Endo implanted tumor from 21.62 +/- 3.55 to 11.32 +/- 2.78 (P < 0.01). CONCLUSIONS: Retroviruses can highly mediate the transfer of endostatin gene into the adenocarcinoma cells. Endostatin gene transfer can inhibit the xenoimplanted tumor growth by its direct inhibition on neovascularization. The combination of endostatin gene transfer with ionizing radiation treatment can synergistically inhibit the neovascularization and the growth of lung adenocarcinoma.

[The expression and clinical implication of gelatinase A in acute leukemic cells].

OBJECTIVE: To study the expression of gelatinase A (MMP2) in acute leukemic cells and its clinical implication. METHODS: The bone marrow samples from 46 acute leukemia patients were investigated by reverse transcription polymerase chain reaction (RT-PCR) and Gelatin Zymography method. Matrigel invasion assay was studied on U937, NB4, K562 and SHI1 cells in vitro. RESULTS: The expression of MMP2 was positive in 24 of 46 patients with AL (52.2%). In MMP2 positive and negative groups of AL, the extramedullary infiltration rates were 50.0% and 18.2% (P < 0.05), whilst the percentage of blast cell in peripheral white blood cells were (77.21 +/- 13.9)% and (62.95 +/- 17.2)% (P < 0.01), respectively. The positive expression of TIMP2 and MT1-MMP in 46 AL patients were 27 (58.7%) and 33 (71.7)%. The matrigel invasion assay suggested that MMP2 is involved in migration of leukemic cell through extracellular matrix (Matrigel). CONCLUSION: The active form of MMP2 might be essential to the egress of leukemic cells from bone marrow into peripheral blood, followed by an extramedullary infiltration.

[Relationship among telomerase activity, expression of human telomerase reverse transcriptase (hTERT), and expression of c-myc in acute leukemia].

BACKGROUND & OBJECTIVE: Regulation of expression of human telomerase reverse transcriptase(hTERT) is regarded as the major determinant of telomerase activity. Several studies suggested that c-myc could regulate hTERT expression. This study was designed to examine the telomerase activity, the expression of hTERT and c-myc, and to analyze their correlations in acute leukemia. METHODS: The telomerase activity was semi-quantitatively assayed in mononuclear cells (MNCs) of bone marrow (BM) from 35 acute leukemia patients and 15 control cases by polymerase chain reaction enzyme- linked immunosorbent assay (PCR-ELISA). The mRNA expression of hTERT and c-myc were determined using reverse transcription polymerase chain reaction (RT-PCR) at the same time. RESULTS: The mean telomerase activity, the expression levels of the hTERT mRNA and c-myc mRNA in acute leukemia were 0.71+/-0.32, 0.47+/-0.36, and 0.85+/-0.31, respectively, which was significantly higher than those of control cases (P< 0.01). The telomerase activity and the expression level of c-myc mRNA were associated with the expression level of hTERT mRNA (P< 0.01). In the 25 follow-up patients, the mean telomerase activity and the expression level of the hTERT were 0.61+/-0.39 and 0.29+/-0.31 in complete remission cases, while 0.83+/-0.23 and 0.53+/-0.31 in none complete remission cases, respectively. There was no significant difference between the two groups. CONCLUSION: The telomerase activity and the expression of hTERT and c-myc mRNA were up-regulated in acute leukemia, and the elevated expression of hTERT may be an important adjuster of telomerase activity. It is likely that c-myc protein can stimulate the expression of hTERT and thereby enhance telomerase activity, which may involve in carcinogenesis of leukemia.

[Molecular biological study of glycoprotein IX gene defect in Bernard-Soulier syndrome].

OBJECTIVE: To identify a mutation G2113-->A in the glycoprotein (GP)IX gene associated with Bernard-Soulier syndrome (BSS) and to investigate BSS pathogenesis. METHODS: Allele-specific restriction enzyme was used to analyze the samples of patient, her mother, her brother and 40 healthy volunteers. Site-directed mutagenesis was performed to construct a expression vector PD-IXG2113A harboring the mutation G2113-->A. Chinese hamster ovary (CHO) cells were transiently cotransfected with plasmids harboring the entire coding region of GPIbalpha, GPIbeta and GPIX or mutant GPIX, respectively. Expression of GPIbalpha and GPIX in transfected CHO cells were analysed with flow cytometer. GPIbalpha and GPIX in the cytoplasma of transfected CHO cells were analysed by immunostaining and Western blotting. RESULTS: The patient was found to be homozygosity of the substitution, her mother and her brother be heterozygous. Expressions of GPIbalpha and GPIX in mutant CHO cells were remarkably reduced, but abundant in the cytoplasma. CONCLUSION: The mutation of Ala139(GCC)-->Thr(ACC) in the GPIX did not affect synthesis and assembly of GPIb/IX complex but influence its anchoring and expression on the cell surface, which was responsible for BSS.

[Establishment of urokinase receptor gene antisense RNA transfer system and its application in leukemia research].

Overexpression of urokinase-type plasminogen activator receptor (uPAR) on tumor cell surface is essential for invasion and metastasis in a variety of tumor cells. To establish a retroviral-mediated antisense RNA transfer system of uPAR gene for exploring its function on down-regulation of uPAR expression in leukemia cells, the retroviral vector LaCD87SN was constructed by inserting uPAR gene into LXSN vector in an antisense orientation. An uPAR gene antisense RNA transfer system was established by liposome-mediated transfection in combination with cross infection with retrovirus. Human leukemia cells U937 were transduced with aCD87 amphotropic retrovirus, expressing uPAR antisense RNA, and the U937/aCD87 cells was obtained by G418 selection. The integration and expression of antisense uPAR gene were analyzed by polymerase chain reaction (PCR) assay. The cell surface expression of CD87 and the activities of matrix metalloproteinase (MMP) were assayed by flow cytometry (FCM) and gelatin zymography, respectively. The results showed that the amphotropic retroviral producers Am12/aCD87, which expressed antisense RNA of uPAR gene with a titer of 6.3 x 10(5) cfu/ml in supernatants, were obtained by means of transfection and superinfection. U937/aCD87 cells were established by continuative G418 selection after retrovirus infection. In U937/aCD87 cells, the integrated provirus and the overexpression of antisense uPAR gene was confirmed. Compared with U937/NeoR cells, FCM analysis revealed that CD87 expression on U937/aCD87 cell surface was not downregulated significantly. However, MMP-9 secretion was significantly suppressed in U937/aCD87 cells. In conclusion, although the retroviral-mediated antisense RNA transfer could not efficiently suppress uPAR expression on leukemic cell surface, it may interfere the uPAR-MMP interactions.

[Electroporation-mediated gene transduction and expression of class 1 aldehyde dehydrogenase (ALDH1) and multidrug resistance gene (MDR1)].

BACKGROUND & OBJECTIVE: This study was designed to seek an efficient and safe method for transfer of genes of large size. METHODS: The retroviral vector containing different kinds drug resistance genes-class 1 aldehyde dehydrogenase (ALDH1) and multidrug resistance gene (MDR1) was linearized by the restriction enzyme NdeI digestion, and introduced into the packaging PA317 cells by electroporation. Selection was performed by vincristine (VCR) and 4-hydroperoxycyclophosphamide(4-HC) to obtain ALDH1 and MDR1 stably expressing cells. The integration of provirus, transcription and translation of foreign genes were confirmed by Southern blot, reverse transcription(RT)-PCR and flow cytometry, respectively. The safety of this delivery system was verified by testing helper virus(envelop gene) using nested PCR. RESULTS: Both ALDH1 and MDR1 were successfully transduced into PA317 cells by electroporation. Stable integration of foreign genes in host cells genome was determined by Southern hybridization blot. The transcription of ALDH1 and MDR1 was demonstrated by RT-PCR. The overexpression of P-glycoprotein encoded by the downstream gene MDR1 with approximately a 4-fold increase in 98% cells was analyzed by flow cytometry. No helper virus can be detected by nested PCR assay. CONCLUSION: These results implicate that the introduction and overexpression of both ALDH1 and MDR1 genes in vitro is attainable by a simple and convenient electroporation method, with the character of safety and high efficiency.

Aldehyde-dehydrogenase gene-transduced hematopoietic cell line K562 overcomes the cytoxicity of cyclophosphamide in vitro.

The identification of genes inducing resistance to anticancer chemotherapeutic agents and their introduction into hematopoietic cells represents a promising approach to overcome bone marrow toxicity, the limiting factor for most high-dose chemotherapy regimens. Because resistance to cyclophosphamide has been correlated with increased levels of expression of the aldehyde-dehydrogenase (ALDH1) gene in tumor cells lines in vitro, this study tested whether ALDH1 overexpression could directly induce cyclophosphamide resistance. Results showed that a retroviral vector was used to transduce full-length human ALDH1 cDNA into human hematopoietic cell line K562 that was then tested for resistance to 4-hydroxycyclophosphamide (4-HC), an active analogue of cyclophosphamide. Overexpression of the ALDH1 gene resulted in a significant increases in cyclophosphamide resistance in transduced K562 cells (50% inhibition concentration, IC50 = 10 micro mol/L). These findings indicate that ALDH1 overexpression is sufficient to induce cyclophosphamide resistance in vitro and provide a basis for testing the efficacy of ALDH1 gene transduction to protect bone marrow cells from high-dose cyclophosphamide in vivo.

[Coexpression of vascular endothelial growth factor and its receptors in human tumor cell lines].

BACKGROUND & OBJECTIVE: Vascular endothelial growth factor (VEGF) play an important role in tumor angiogenesis through a paracrine mechanism, but the importance of its autocrine has not been fully elucidated. This study was designed to explore the gene coexpression pattern of VEGF and its receptors (Flt-1 and KDR) in variety of human malignant cell lines. METHODS: After isolation of RNA from thirty tumor cell lines and four endothelial cells, gene expressions of VEGF and its receptors were measured by semi-quantitative reverse transcriptase polymerase chain reaction using the transcriptional level of the house-keeping gene for internal calibration. RESULTS: VEGF transcript was detected in the majority of tumor cell lines (29/30) and in all endothelial cell lines at moderate or high level, while the expression of VEGF was low in human umbilical vein endothelial cell (HUVEC). Moreover, Flt-1 gene expression was found in 50% of hematopoietic malignancies (6/12), 28% of solid tumors (5/18), and endothelial cells (EA. hy926 and HUVEC). In contrast, the expression of KDR gene was found in 2 (16.7%) hematopoietic cell lines and 6 (33.3%) solid tumor lines. In endothelial cells, KDR gene expression was detectable in HMEC-1 and HUVEC only. However, ECV304 cells express neither Flt-1 nor KDR gene. CONCLUSION: Overexpression of VEGF gene in all tumor cell lines provides a potential biological marker for malignancies and the coexpression of VEGF with its receptors suggesting an autocrine pathway exists in tumor cells.