AIM The hepatic content of collagens (type I, Ⅲ and Ⅶ) and laminin (LN) in rat model of experimentalliver fibrosis was observed to find out their roles in the pathogenesis of liver fibrosis.METHODS The experimental rat model was established by immunological injury induced by injectinghuman albumin. Histopathological and immunohistochemical methods were used to measure the hepaticcontent of collagens and laminin in the fibrotic rat livers.RESULTS The hepatic contents of collagens (type I, Ⅲ, Ⅶ) and LN in the fibrotic rat livers weresignificantly increased as compared with those in the control group, and they were found to be mainlylocalized in the portal space, central veins and fibrous septa. Electron microscopic study showed that pro-collagens were present around the “activated” hepatic stellate cells (HSC) and the hepatocytes atrophied.CONCLUSION Pathological deposition of collagens (type Ⅰ, Ⅲ and Ⅶ ) and laminin was the fundamentallesion of liver fibrosis. HSC may be the major cellular source of collagens (type Ⅰ, Ⅲ and Ⅶ) and laminin inthe liver tissue.

AIM To investigate the effects of collagen solution on the prevention of acute gastric mucosal injury inrestricted rats inflicted by cooling in low temperature (4℃),METHODS Thirty healthy Wistar rats were randomly divided into normal (N, n = 10),injury (I, n = 10)and prevention (P, n = 10) groups. The rats were fasted for 48 h but free access to water without restrictionand cooling in normal group, fasted for 48 h but free access to water with restriction of rats onto the fixationframe for cooling in 4℃ for 4 h, so to cause stress injury of gastric mucosal membrane in I group and fed with3 mL of collagen solution 30min before injury in P group in addition to the procedures in I grobp. Gastricmucosal potential difference, blood flow volume, content of nitrite (NO2-) and hydrogen ion concentration(H+ ) in gastric juice were determined under aneasthesia at 48 h after fast in N group and at 4 h after injuryin I and P groups to evaluate the degree of injury (injury index).RESULTS Gastric mucosal potential difference was 22.10±5.27 in N group and 11.46±5.25 in I groupwith obvious difference (P<0.01), but 16.98±4.84 in P group which was remarkably improved whencompared to that in I group. Gastric mucosal blood flow volume was 23.65±10.65 in I group and 57.20±11.75 in N group with evident difference (P<0.01), but 37.49±5.87 in P group with sound effects incontrast to that in I group (P<0.01). Gastric injury index was 18.40±8.35 in I group and 7.9±2.13 in Pgroup with significant difference (P<0.01). Hydrogenion concentration in gastric juice was 118.0±41.2mmol/L in N group, 186.9±74.7 mmol/L in I group and 96.4±57.2 mmol/L in P group with prominentdifference (P< 0.01 ) between those in I and P group. Gastric mucosal nitrite concentration was 1.15±0.46in N group, 0.69±0.15 in I group and 1.04±0.44 in P group with obvious differences between N and Igroups (P<0.01) and between I and P group (P<0.01).CONCLUSION Ischemic and hypoxic injury of gastric mucosal due to low blood perfusion during restrictionand cooling injury at 4℃ was supposed to be an important factor in inducing gastric mucosal stress injury. Butcollagen solution could maintain the integrity of gastric mucosal barrier, buffer gastric acid, promotethrombocytic agglutination and ameliorate direct injury to gastric mucosa caused by various factors.

A novel double-layer collagen membrane with unequal pore sizes in each layer was designed and tested in this study. The inner, loose layer has about 100-μm-diameter pores, while the outer, compact layer has about 10-μm-diameter pores. In a rat model of incomplete spinal cord injury, a large number of neural stem cells were seeded into the loose layer, which was then adhered to the injured side, and the compact layer was placed against the lateral side. The results showed that the transplantation of neural stem cells in a double-layer collagen membrane with unequal pore sizes promoted the differentiation of neural stem cells, attenuated the pathological lesion, and signiifcantly improved the motor function of the rats with incomplete spinal cord injuries. These experimental ifndings suggest that the transplantation of neural stem cells in a double-lay-er collagen membrane with unequal pore sizes is an effective therapeutic strategy to repair an injured spinal cord.

We previously showed that the repair of bone defects is regulated by neural and vascular signals. In the present study, we examined the effect of topically appliedβ-nerve growth factor (β-NGF) on neurogenesis and angiogenesis in critical-sized bone defects iflled with collagen bone substi-tute. We created two symmetrical defects, 2.5 mm in diameter, on either side of the parietal bone of the skull, and filled them with bone substitute. Subcutaneously implanted osmotic pumps were used to infuse 10 μgβ-NGF in PBS (β-NGF + PBS) into the right-hand side defect, and PBS into the left (control) defect, over the 7 days following surgery. Immunohistochemical staining and hematoxylin-eosin staining were carried out at 3, 7, 14, 21 and 28 days postoperatively. On day 7, expression of β III-tubulin was lower on theβ-NGF + PBS side than on the control side, and that of neuroiflament 160 was greater. On day 14,β III-tubulin and protein gene product 9.5 were greater on theβ-NGF + PBS side than on the control side. Vascular endothelial growth factor expression was greater on the experimental side than the control side at 7 days, and vascular endothelial growth factor receptor 2 expression was elevated on days 14 and 21, but lower than control levels on day 28. However, no difference in the number of blood vessels was observed between sides. Our results indicate that topical application ofβ-NGF promoted neu-rogenesis, and may modulate angiogenesis by promoting nerve regeneration in collagen bone substitute-iflled defects.

脉冲掺钕钇铝石榴石激光对增生性瘢痕成纤维细胞胶原合成的影响

Objective In order to explore the selectively inhibitory effects of pulsed Nd:YAG laser irradiation on collagen production of scar fibroblasts in vitro.Methods Cultured fibroblasts derived from hypertrophic scars(HS) and normal human skin were irradiated with a pulsed Nd:YAG laser(wavelength 1 064 nm,pulse width 150 μ s) at various energy density levels (500,1 000,1 500 and 2 000 J/cm2).At 24 hours after laser irradiation, collagen production of fibroblasts was measured by the incorporation of 3H proline. The expression of proα 1(I)procollagen mRNA was investigated by blot hybridization techniques. Results Collagen production of HS fibroblasts was significantly increased, as 2 times as that of normal skin fibroblasts. Type I procollagen mRNA level in HS fibroblasts was markedly elevated, as 3 times as that in normal skin fibroblasts.Conclusion Pulsed Nd:YAG laser at energy density of 1 000 J/cm2 can selectively suppress collagen synthesis and type I procollagen mRNA level of HS fibroblasts.

恒河猴异体椎间盘移植组织学活性和生物化学研究

Objective To investigate the histological viability and the biochemical change of fresh allografted intervertebral disc of rhesus monkey.Methods Under general anaesthesia,osteotomies of 12 rhesus monkeys were made at the endplates about 2.0 mm away from the L4～ 5 intervertibral disc,then the fresh discs were transposed in two matched monkeys. The four groups of animals were sampled after the third, sixth,ninth and twelfth month respectively.Histological viability was assessed by 3H Proline incorporation and biochemical changes were measured by water,proteoglycan and collagen content. Results Study of histological viability demonstrated that the DPM (Disintegration Per Minute) of 3H Proline increased at the third month in anulus fibrous and the sixth month in nucleous pulposus.In the nucleus pulposus of implanted disc the study of biochemistry showed that proteoglycan content was not change obviously nine months ago, but proteoglycan content decreased at the twelfth month.Collagen contents increased at the third,sixth and ninth month. Water contents were not change among twelve months. In the annulus fibrous of implanted disc,proteoglycan and water contents decreased at the ninth month, but they were increased at the twelfth month. Collagen content increased at the sixth month,but it decreased at the ninth month again. Conclusions The present results showed the intervertibral disc grafted survived allologous transplantation.The fresh allografted intervertibral disc appeared some ability of regeneration.

兔耳增生性瘢痕的形态学观察与羟脯氨酸含量变化

Objective To establish animal model for hypertreophic scar and study the characteristics of its morphology and collagen metabolism.Methods A total of 64 wounds(diameter of 6 mm each ) with total skin loss were made on the ventral side of rabbit ear using a trephine. Morphology and collagen metabolism of scar wound were studied at 14,21,35,70 and 98 days after operation,respectively.Results There were 76% elevated scars developed (45/59 wounds) on the ventral side of rabbit ear on 21st and 35 days.The number of fibroblast decreased,but irregular-arranged fibers still presented in the elevated scars at 70 and 98 days after operation. Hydroxyproline content in elevated scars at 21 days was higher than that in normal skin(P< 0.05),and at 35 days was 3 times as that in normal skin and at 98 days was also markedly higher than that in normal skin(P< 0.05).Conclusion Excessive deposition of collagen is a characteristic of hypertrophic scar in rabbits. The conversion of normal scarring to hypertrophic scarring in rabbits occurs at 14～ 21 days after operation. Both development and regression of hypertrophic scar in rabbit are quicker than that in human.

Objective To investigate the effects of dexamethasone on the promoter activity of human al(1) procollagen gene.Methods Fibroblasts from human skin were primary cultured and subcultured. (1) The effects of dexamethasone on the human skin fibroblasts were determined by BrdU incorporation into DNA of fibroblasts. (2) Three plasmids containing various engths of 5＇ flanksequence of human al(1) procollagen gene and CAT as reporter gene were constructed, and were transfected into the human skin fi-broblasts by FuGENE Transfecfion Reagent. The effects of dexamethasone on 3 plasmids were determined by CAT - ELlSA. Results (1)After 24h of treatment on the fibroblasts with 110-9 ～110-4mol/L examethasone in DMEM containing 2% or 10% FCS, BrdU in-corporation into DNA showed no difference ( P > 0.05) . (2) the 3 plasmids were transfected into fibroblasts and then treated with 110-5mol/L and 110-6mol/L dexamethasones for 24h, relative CAT values were different belwent dexamethasone and control,higher dexamethasone(110 -5mol/L} and lower examethasone(110 -6mol/L) ( P <0. 05) . ConcluSion Dexamethasone has noeffects on the proliferation of human skin fibroblasts, and it has negative effect on the promoter activity of human al(1) procollagengene, which is dose- dependent.

Objective: To culture fibroblast cells from the kneeligaments and to study the biological characteristics of thesecells.Methods: Cells of the anterior cruciate ligament(ACL) and the medial collateral ligament (MCL) fromNew Zealand white rabbit were cultured in vitro. Cellulargrowth and expression of the collagen were analyzed.Moreover, an in vitro wound closure model was establishedand the healing of the ACL and the MCL cells wascompared.Results: Maximal growth for all these cells wereobtained with Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum, but RPMI 1640and Ham's F12 media were not suitable to maintain thesecells. Morphology of both ACL and MCL cells from NewZealand white rabbit was alike in vitro, but the MCL cellsgrew faster than the ACL cells. Both cell types producedsimilar amount of collagen in culture, but the ratio ofcollage type I to type III produced by ACL cells was higherthan that produced by MCL cells. Wound closure assayshowed that at 36 hours after injury, cell-free zones createdin the ACL cultures were occupied partially by the ACLcells; in contrast, the wounded zone in the MCL cultureswas almost completely covered by the cells.Conclusions: Although the ACL cells and the MCLcells from New Zealand white rabbit show similarappearance in morphology in culture, the cellular growthand the biochemical synthesis of collagen as well as thehealing in vitro were significantly different. Thesedifferences in intrinsic properties of the two types of cells invitro might contribute to the differential healing potentialsof these ligaments in vivo.

Objective: To study the influence of stress-relaxation plate on disorganization and repair of the cortex beneath the plate.Methods: A washer made of viscoelastic polyethylene was placed between the screw and the screw hole of conventional stainless rigid plate (RP) to produce a stressrelaxation plate (SRP). Both SRP and RP were applied to osteotomized tibia in 48 New Zealand rabbits. Healing process of the fracture with either SRP or RP fixation (control) was comparatively studied with polarized light microscopy, in situ hybridization of collagen mRNA and immunohistochemical technique from 2 to 36 weeks postoperatively.Results: The study of plated bone remodeling showed that the degree of cortex osteoporosis beneath the plate was similar between the SRP and RP group within 12 weeks postoperatively. In comparison, the disorganization of bone structure in SRP group happened later and milder than that of RP group, and the repair process began at 12 weeks after implantation. As a consequence, the absorption cavities became smaller and the structure of collagen fibers became well oriented along with these changes by polarized light microscopy. In addition to these, the in situ hybridization analysis of collagen genes and the immunohistochemical study of type Ⅰ , Ⅲ collagen showed that the osteoblasts lying on the surface of absorption cavities expressed and synthesized type Ⅰ collagen at 8 to 12 weeks after implantation. From this time on, the changes above became more evident significantly before most of cavities were repaired by 36 weeks. In contrast to the changes in the SRP group, no expression and synthesis of any kind of collagen could be observed during 12 to 36 weeks after implantation in RP group.Conclusions: Without removal of the bone plate, the SRP fixation not only reduces the degree of plated bone osteoporosis, but also makes the disorganized bone structure restored to normal in terms of the expression and synthesis of type Ⅰ collagen mRNA of osteoblasts lying on the surface of absorption cavities.