[Multi-scenario Simulation of Construction Land Expansion and Its Impact on Ecosystem Carbon Storage in Beijing-Tianjin-Hebei Urban Agglomeration].

It is of great practical significance for regional sustainable development and ecological construction to quantitatively analyze the impact of construction land expansion on terrestrial ecosystem carbon storage and to explore the optimization scheme of simulating construction land expansion to improve future ecosystem carbon storage. Based on the land use and cover change (LUCC) and other geospatial data of the Beijing-Tianjin-Hebei Urban Agglomeration from 2000 to 2020, this study utilized the Integrated Valuation of Ecosystem Services and Tradeoffs (InVEST) model and the patch-generating land-use simulation (PLUS) model to assess and analyze the changes in ecosystem carbon stocks and spatial patterns regionally. In this study, we performed linear regression analysis to investigate the relationship between urban land expansion and changes in ecosystem carbon stocks for varying urban land proportion levels during two distinct time intervals, 2000-2010 and 2010-2020, which was conducted at a spatial resolution of 2 km. Three distinct urban land expansion scenarios were subjected to simulation to forecast the prospective land use pattern by 2030. Subsequently, we quantified the ramifications of these scenarios on ecosystem carbon stocks during the period from 2020 to 2030. The results were as follows:① In the Beijing-Tianjin-Hebei Urban Agglomeration, the ecosystem carbon stocks exhibited notable variations over the study period, with values of 2 088.02, 2 106.78, and 2 121.25 Tg recorded for the years 2000, 2010, and 2020, respectively, resulting in a cumulative carbon sequestration of 33.23 Tg C during the study duration. It is noteworthy that forest carbon storage emerged as the dominant contributor, with an increase from 1 010.17 Tg in 2000 to 1 136.53 Tg in 2020. Throughout the study period, the spatial distribution of carbon stocks displayed relative stability. Regions characterized by lower carbon content were concentrated in the vicinity of the Bohai Rim region and in proximity to cities such as Beijing, Tianjin, and Shijiazhuang, as well as rural settlements. In contrast, grid units with moderate and high carbon stocks were predominantly situated in the western Taihang Mountain and the northern Yanshan Mountain. Additionally, there was a tendency of increasing carbon stocks in the Taihang Mountain and Yanshan Mountain region, whereas those surrounding major urban centers such as Beijing, Tianjin, Shijiazhuang, and Tangshan experienced a notable decline in carbon stocks. Such reductions were most pronounced in regions undergoing urban land expansion during the study period. ② In grid units with an urban land proportion exceeding 10% at each level, a strong correlation was observed between urban land expansion and changes in carbon stocks during both the 2000-2010 and 2010-2020 periods. The changes in urban land proportion adequately explained the variations in carbon stocks. However, the explanatory power of urban land on carbon stocks decreased during the 2010-2020 period, indicating that other factors played a more substantial role in influencing carbon stocks during this time. The regression coefficients for both periods exhibited a fluctuating upward trend. In comparison to that during the 2000-2010 period, the impact of urban land expansion on carbon stocks was relatively smaller during 2010-2020, indicating a weakening influence. ③ In light of three distinct development scenarios, namely natural development (Scenario Ⅰ), a 15% reduction in the rate of urban land expansion (Scenario Ⅱ), and a 30% reduction in the rate of urban land expansion (Scenario Ⅲ), the projected ecosystem carbon stocks for the Beijing-Tianjin-Hebei Urban Agglomeration in the year 2030 were estimated to be 2 129.12, 2 133.55, and 2 139.10 Tg, respectively. These projections indicated an increase of 7.88, 12.30, and 17.85 Tg in comparison to the current carbon stocks. All scenarios demonstrated that the terrestrial ecosystem would play a role of carbon sink, particularly with the greatest carbon sink observed in the scenario with a 30% reduction in urban land expansion. The fit performance between urban land expansion and carbon stock changes during the 2020-2030 period was significantly better than that during the 2000-2010 and 2010-2020 periods, and the regression coefficients showed a fluctuating increase with an increase in urban land proportion. Across grid units with different urban land proportion levels, the regression coefficients exhibited the order of Scenario Ⅰ < Scenario Ⅱ < Scenario Ⅲ. In pursuit of the carbon peaking and carbon neutrality goals, the Beijing-Tianjin-Hebei Urban Agglomeration should prioritize scenarios with reduced rates of urban land expansion, especially in regions with higher urban land proportions.

Identification of three novel SRD5A2 mutations in Chinese patients with 5α-reductase 2 deficiency.

In this study, we investigated the genetics, clinical features, and therapeutic approach of 14 patients with 5α-reductase deficiency in China. Genotyping analysis was performed by direct sequencing of PCR products of the steroid 5α-reductase type 2 gene (SRD5A2). The 5α-reductase activities of three novel mutations were investigated by mutagenesis and an in vitro transfection assay. Most patients presented with a microphallus, variable degrees of hypospadias, and cryptorchidism. Eight of 14 patients (57.1%) were initially reared as females and changed their social gender from female to male after puberty. Nine mutations were identified in the 14 patients. p.G203S, p.Q6X, and p.R227Q were the most prevalent mutations. Three mutations (p.K35N, p.H162P, and p.Y136X) have not been reported previously. The nonsense mutation p.Y136X abolished enzymatic activity, whereas p.K35N and p.H162P retained partial enzymatic activity. Topical administration of dihydrotestosterone during infancy or early childhood combined with hypospadia repair surgery had good therapeutic results. In conclusion, we expand the mutation profile of SRD5A2 in the Chinese population. A rational clinical approach to this disorder requires early and accurate diagnosis, especially genetic diagnosis.

Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters.

BACKGROUND: We have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells. RESULTS: Sixty-one single-cell-derived dermal fibroblast clones were established from human foreskin by limiting dilution culture. Of these, two clones could be differentiated into neuron-, adipocyte- or hepatocyte-like cells under certain culture conditions. In addition, those two clones were able to differentiate into islet-like clusters under pancreatic induction. Insulin, glucagon and somatostatin were detectable at the mRNA and protein levels after induction. Moreover, the islet-like clusters could release insulin in response to glucose in vitro. CONCLUSIONS: This is the first study to demonstrate that dermal fibroblasts can differentiate into insulin-producing cells without genetic manipulation. This may offer a safer cell source for future stem cell-based therapies.

A splice site mutation combined with a novel missense mutation of LHCGR cause male pseudohermaphroditism.

Leydig cell hypoplasia (LCH) is a rare form of male pseudohermaphroditism caused by inactivating mutations in the luteinizing hormone receptor gene (LHCGR). The majority of LHCGR mutations are located in the coding sequence, resulting in impairment of either LH/CG binding or signal transduction. We report a Chinese family with two siblings (46, XY and 46, XX) carrying a missense mutation (c. 455 T>C, p. Ile152Thr) and a splice site mutation (c. 537-3 C>A). Computational analysis of the missense mutation in the three-dimensional structural model predicted it might influence the distribution of hydrogen bonds and intermolecular contacts between the hormone and receptor. Consistent with these findings, in vitro mutant analysis revealed a marked impairment of human chorionic gonadotropin binding and signal transduction. The splice-acceptor mutation (c. 537-3 C>A) resulted in abnormal splicing of LHCGR mRNA, skipping exon 7. This report expands the genotypic spectrum of LHCGR mutations, with relevant implications for the molecular analysis of this gene.

[An experimental study on influence of concentration of fetal bovine serum on chondrogenesis of BMSCs].

OBJECTIVE: To explore the chondrogenetic effect of induce media containing different concentrations of fetal bovine serum (FBS) on BMSCs differentiation in vitro and provide technical parameters for cartilage engineering in vitro. METHODS: Passage 2 BMSCs of swine were seeded at the density of 5 x 10(7) cells/cm3 to disc-shaped PGA scaffolds with a diameter of 5mm and a thickness of 2mm. After 7days, the scaffolds were induced in media with TGF-beta1, IGF-I, dexamethasone, and different concentrations of FBS: 0% in A group, 5% in B group, and 10% in C group. Specimens were collected after 8 weeks for gross observation, size evaluation, wet weight, glycosaminoglycan (GAG) content, histology assessment, and immunohistology of type II collagen. RESULTS: The compound of C group showed china-white color, hard and fine texture, no obvious change in size and shape, typical lacuna structures, cartilage specific ECM, and significantly higher wet weight and GAG content. The compound of B group showed reduced size, fewer lacuna structures and some cartilage specific ECM. And the compound of A group showed greatly reduced size, soft and loose texture, and no typical lacuna structure or cartilage specific ECM. CONCLUSIONS: FBS was indispensable to chondrogenetic media for in-vitro tissue engineering of cartilage with BMSCs.

[Influence of transforming growth factor-beta1 inducing time on chondrogenesis of bone marrow stromal cells (BMSCs): in vitro experiment with porcine BMSCs].

OBJECTIVE: To explore the influence of transforming growth factor (TGF)-beta1 inducing time on the chondrogenesis of bone marrow stromal cells (BMSC), and on the construction of tissue engineering cartilage. METHODS: BMSCs were obtained from the greater trochanters of 3 pigs, cultured, seeded onto the cylindrical scaffolds made of polyglycolic acid at the density of 5.0 x 10(7) cells/ml, and then cultured with chondrogenesis media containing TGF-beta(1) (10 ng/ml), insulin-like growth factor-I (50 microg/L), and dexamethasone (40 microg/L) to be induced by TGF-beta(1) for 2 weeks (Group A), 4 weeks (Group B), 6 weeks (Group C), 8 weeks (Group D), or 10 weeks (Group E) respectively. 10 weeks later the cylindrical scaffolds underwent gross observation and histological examination. Alcin blue method was used to examine the content of proteoglycan (GAG). Immunochemistry and Western blotting were used to examine the type II collagen. The cylindrical scaffolds underwent biomechanical analysis. RESULTS: HE staining showed cartilage lacunae increasing in number from the periphery to center of the cylindrical scaffolds with the extended inducing time, and were arranged more uniformly progressively. Histochemical staining showed GAG accumulation. Immunohistochemistry and Western blotting showed that the content of type II collagen increased gradually, the amount of type II collagen stabilized after 6 weeks' culture, and there was no significant difference in the content of type II collagen between Group C and Group E. Biomechanical analysis showed that the modulus of elasticity and compression strength of the groups induced for more than 6 weeks (Groups C, D, and E) were all higher then those of Groups and B. CONCLUSION: TGF-beta(1) inducing time is correlated with the cartilage engineering characteristics with BMSC as seed cells. Induction for 6 weeks helps construct tissue engineered cartilage with good tissue structure, chemical composition and biomechanical properties.

Clonal analysis of nestin(-) vimentin(+) multipotent fibroblasts isolated from human dermis.

Although several studies have shown that dermal fibroblasts possess adipogenic, osteogenic or chondrogenic differentiation potential, no study has characterized this cell population in detail, and there is as yet no evidence that a single dermal fibroblast can differentiate into all these types of cells. In this study, dermal fibroblasts were isolated from human foreskin using a regular dermal fibroblast culture system. These cells could be expended in adherent culture for over 40 cell doublings. In addition, dermal fibroblasts exhibited adipogenic, osteogenic and chondrogenic phenotypes when they were cultured in the presence of certain inducers. Importantly, clonal analysis showed that 6.4% (3/47) of the single-cell-derived clones were tripotent, 19.1% (9/47) of the clones were bipotent, and 10.6% (5/47) of the clones were unipotent. Furthermore, one of the three tested tripotent clones exhibited neurogenic and hepatogenic differentiation potential. Phenotypic analyses showed that the tripotent fibroblasts were nestin(-) vimentin(+), which is different from the dermis-derived stem cells reported by others. These results indicate that dermal fibroblasts are a heterogeneous population containing progenitors with various levels of differentiation potential, and the nestin(-) vimentin(+) fibroblasts may represent a novel type of multipotent adult stem cells in human dermis.

[Effect of adeno-BMP7 transfection on osteogenesis of BMSCs].

OBJECTIVE: To study the effect of adeno-BMP7 transfection on the biology of bone marrow stromal cells (BMSCs). METHODS: Bone marrow was obtained from the goat. The BMSCs were isolated and cultured at the second passage. Once the cells attached and formed a monolayer with 70%-80% confluency, adeno-BMP7 (M.O.I. = 100) was added to the cells. After three days, calcium node was examined with staining; cell-coral compound was replanted subcutaneously. RESULTS: With adeno-BMP7 transfection, BMP7 expression was detected with Western-blot; big calcium nodes were observed with staining. New bone formation was enhanced, which was evaluated by X-ray and histological examinations. CONCLUSIONS: BMSCs transfected with adeno-BMP7 show much stronger osteogenic ability.

[Cartilage-derived morphogenetic protein 1 initiates chondrogenic differentiation of human dermal fibroblasts in vitro].

OBJECTIVE: To explore the factors influencing the differentiation of fibroblasts into chondrocyte phenotype induced by a growth factor, cartilage-derived morphogenetic protein 1 (CDMP1). METHODS: Fibroblasts isolated from foreskin obtained during circumcision were cultured in the forms of micromass and monolayer culture. The culture fluid of the fibroblasts at the passage 2, 7, and 10 was added with CDMP1 of the concentrations at the concentrations of 10, 30, 100, and 300 ng/ml respectively and co-cultured for 7 days. RT-PCR was used to detect the expression of bone morphogenetic protein receptor (BMPR), activin receptor-like kinase (ALK) receptor, collagen types II, IV, and X before and after CDMP1 induction. Western blotting was used to detect the protein expression of collagen types II, a transcriptional factor Sox9, and aggrcan before and after the induction. Flow cytometry was used to detect the superficial markers CD29, CD105, CD106, and CD166, and the expression of collagen types I and II. RESULTS: Western blotting showed that the collagen type II positive cell rate in the passage 5 cells was 74.3% +/- 0.4%, not significantly different from that of the passage 2 cells (73.4% + 0.5%). When the concentrations of CDMP1 were 10 and 30 ng/ml no expression of aggrecan and collagen type II was detected, When the concentrations of CDMP1 was 100 and 300 ng/ml, the expression of aggrecan and collagen type II could be detected and without significant differences between these 2 concentrations. The expression of aggrcan and collagen type II mRNA disappeared in the monolayer cultured P2 and P5 fibroblasts induced by CDMP1 for 14 days, However, RT-PCR and Western blotting showed expression of collagen type II, aggrcan and SOX9 in the micromass cultured fibroblasts. RT-PCR showed that all fibroblasts cultured in vitro expressed ActR-I/ALK-2, BMPR-IA/ALK-3, and BMPR-IB/ALK-6 genes, and the expression of these genes significantly increased after CDMP1 for 7 days. CONCLUSION: CDMP1 stimulates the human dermal fibroblasts expanded in vitro to differentiate into chondrogenic phenotype in a dose dependent manner. Three-dimensional culture environments accelerate the chondrogenic differentiation. The expression of ALK receptors may involve the CDMP1 stimulated differentiation of fibroblasts.

[Cartilage-derived morphogenetic protein 1 initiates chondrogenic differentiation of human dermal fibroblasts in vitro].

OBJECTIVE: To investigate the feasibility of human dermal fibroblasts in vitro differentiation into chondrogenic phenotype with induction of cartilage-derived morphogenetic protein (CDMP) growth factor. METHODS: Human dermal fibroblasts were isolated from foreskin and cultured in monolayer ex vivo. Dermal fibroblasts of passage2 was plated at density of 1 x 10(4) cells/cm(2) and induced with CDMP1 (100 ng/ml) in medium of F12 + 10% FBS. After 7 days of induction, morphology of cells was observed under phase-contrast microscopy and the length:width ratio of cells was calculated by Image Plus software analysis. Expression of type I, II, III collagen was detected by immunofluorescence and observed with confocal microscopy. The method of Western-Blot was applied to detect secretion of collagen type II. mRNA expression of chondrogenic related Sox9, Aggrecan as well as collagen type II, IX was detected by RT-PCR. The osteogenic related expression of collagen type X, Alkaline Phosphatase (AKP) was also detected by RT-PCR. Pellet cultured dermal fibroblasts at a density of 2 x 10(7) cells/ml was observed respectively for proteoglycan and collagen type II expression with Alcian blue and immunohistochemistry staining. RESULTS: With the induction of CDMP1, the morphology of cells changed from spindle fibroblastic appearance to that of typical chondrocyte-like polygon shape. By Image Plus software analysis, it was found that the length/width ratio changed significantly from 7.40 +/- 1.30 of preinduction to 1.40 +/- 0.15 of post-induction (P < 0.05). No significant difference was found between the postinduction and normal chondrocyte (1.29 +/- 0.24). By confocal microscope observation, expression of collagen type II was found intracellularly in CDMP1 treated fibroblasts. Western-Blot detection confirmed collagen type II expression by 7 days induction. RT-PCR gene expression analysis of characteristic chondrogenic related genes, such as Sox9, Aggrecan as well as collagen type II, IX, revealed induction of chondrocytic phenotype in monolayered culture upon stimulation with CDMP1 for 7 days. While osteogenic related gene expression of collagen type X, AKP was not detected by RT-PCR, which indicates that osteogenic differentiation was not initiated by CDMP1 in 7 days culture. Histological staining of proteoglycan with Alcian blue and immunohistochemical staining cartilage specific type II collagen revealed deposition of typical cartilage extracellular matrix deposition in pellet cultured fibroblasts. CONCLUSION: These results suggests human dermal derived fibroblast could be differentiated into chondrogenic phenotype with CDMP1 induction in vitro.

[Influence of mechanical stress on chondrogenesis of in vitro cultured porcine bone marrow stem cells: a preliminary study].

OBJECTIVE: To evaluate the influence of mechanical stress on chondrogenesis of in vitro cultured porcine bone marrow stem cells (BMSC). METHODS: Porcine BMSC of passage 2 were seeded onto a cylinder-shaped PGA/PLA scaffold, 8mm in diameter and 3mm in thickness, at a density of 5 x 10(7)/cm(3). After the cell-scaffold constructs were cultured for one week, the primary medium, high-glucose DMEM medium with 10% fetal bovine serum (FBS), was replaced by chondrogenically inductive medium containing TGFbeta(1) (10 ng/ml), IGF-I (50 ng/ml), and dexamethasone (40 ng/ml) in addition to DMEM+10% FBS. The constructs were randomly divided into three groups according to the imposed stress: experimental group A in which a centrifugal stress was imposed at 100 g, 30 min, 2/d; experimental group B in which a rotative stress was imposed at 80 rpm, 8 h/d by a shaker; and control group in which the constructs were statically cultured. The gross view, histology, histochemistry, immunohistochemistry and glycosaminoglycan (GAG) content were evaluated after 4 and 8 weeks respectively. RESULTS: Four weeks later, the constructs in both experimental groups maintained their original sizes and shapes. Histology showed nodular lacuna-like structures, in company with GAG deposition and collagen synthesis. In addition, collagen type II was detected by immunohistochemistry. In the control group, however, the constructs shrunk to a little smaller size than those in the experimental groups, and histological staining showed a little amount of lacuna. Eight weeks later, the constructs in both experimental groups still maintained the original sizes and shapes with good elasticity. HE staining showed massive lacuna-like structures in most areas of the construct and extracellular matrix deposited evenly. Fibrous tissues were only observed in some areas. Safranin-O staining showed massive GAG formation and Masson staining showed much more collagen formation than those in the control group. Immunohistochemical staining of collagen type II showed strong positive expression. In the control group the constructs showed massive fibrous tissues, with a small amount of lacuna-like structures in the peripheral areas. GAG contents in the 2 experimental groups were 5.98 mg/g and 5.62 mg/g respectively, both significantly higher than that in the control group (4.73 mg/g) without a difference between the 2 experimental groups. CONCLUSION: Mechanical stress promotes chondrogenesis and cartilage maturation of BMSC in vitro.

[Experimental study of porous TCP to generate tissue-engineered long bone].

To study bone-forming of a new kind of porous beta-TCP as the scaffold for tissue-engineering, defects at the mid-portion of the left and right ulna were created in dog, the defects were repaired with beta-TCP cylinder coated with BMSCs, and beta-TCP cylinders alone as control. X-rays showed the defects were better bridged by the replant with obscure edge and new bone formed in the canal and at the interface in experimental group after three month of operation, whereas in control group, the replants were obviously deformed into dissociated granule with unequal density with only little new bone formed at the interface. After six month, the defects were bridged by new bone with osteodermatous cavum medullare ossium, but in control group, the defects were bridged by high density in radiography without osteodermatous cavum medullare ossium, the diameter of the ular was obviously less than experimental group. There were significant differences between both groups at month one and two in the development pattern through radionuclide observation. By gross, the diameter of ular was smaller in control group than in experimental at month three, and the replants in control group was difficult to detach from the fibroid tissue around it, but in experimental group, there was much more new bone formation, and the surface was rough for the compound of new bone and beta-TCP undegraded completely. The new bone in experiment had been obviously remodeled at month six, but at this moment, the new bone was of infirmity in volume and form. HE staining of three months demonstrated new bone adhered to the surface on the core of beta-TCP in experimental group, but in control group, at the same place, osteoid was observed with much megacayocytes and capillaries. At month six, beta-TCP disappeared completely with new bone formed in both groups, but the volume and structure of the bone was better in experimental group than in control group. From this study it is concluded that the porous beta-TCP can be combined with BMSCs, and the combination could generate new bone to repair long bone defect.