BACKGROUND: Nerve growth factor (NGF) is characterized by poor stability both in vitro and in vivo, and liable to lose its bioactivity.OBJECTIVE: To study the stability of liposome-encapsulated NGF injection preserved under various conditions.DESIGN: A controlled study of liposome-encapsulated NGF.SETTING: Department of Surgery of the First Affiliated Hospital of Nanjing Medical University.MATERIALS: This study was carried out at the Central Laboratory of the First Affiliated Hospital of Nanjing Medical University between July 2002and March 2004. NGF from rat submaxillary gland was purified, encapsulated by liposome, and prepared into lyophilized dosage form before preserved under different conditions (at 4 ℃, room temperature, 40 ℃ or 40 ℃ with saturated humidity, respectively).METHODS: Chicken embryo dorsal root ganglion cultured in serum-free medium was used to evaluation the bioactivity of NGF in vitro. The dorsal root ganglion from 8-day-old chicken embryo was inoculated in a polylysine-coated 24-well culture plate and cultured in Dulbecco modified Ea-gle medium (DMEM) containing different testing samples. Only DMEM was used for culture in the negative control group, while DMEM containing NGF at different concentrations used in the positive control group. The ganglion was cultured at 37 ℃ with 50 mL/L CO2 and saturated humidity for 24 hours, and the growth of the nerve fibers was observed under an inverted microscope. The bioactivity of NGF was also evaluated in simulated condition in vivo by adding lyophilized liposome-encapsulated NGF and positive control NGF specimen into 0.5 mL rat serum, which, along with the blank control serum, was added into 2.5 mL DMEM at 0.5, 1, 2, 3, 4,6 hours and thoroughly mixed. The bioactivity of NGF was assessed accord-ing to the length and density of the dorsal root ganglion and graded the prominences (recorded as "++" or "+++ "), and very long and dense growth of the prominences (++++).MAIN OUTCOME MEASURES: In vitro bioactivity of NGF preserved for 10, 30, 60, and 90 days by testing the growth of cultured chick embryo dorsal root ganglion in serum-free DMEM and in test of rats serum containing NGF added at 0, 0.5, 1, 2, 3, 4, and 6 hours into DMEM.RESULTS: Lyophilized liposome-encapsulated NGF exhibited stable bioactivity (+++) after preservation at 4℃ and room temperature for 10-90days; at 40 ℃ for 10-60 days, the it retained its the bioactivity (+++),which, however, slightly decreased by 90 days (++); its bioactivity was preserved (+++) at 40 ℃ with saturated humidity for 10 days (+++), slightly decreased at 30-60 days (++) and noticeably lowered (+) at 90 days. When preserved for 0, 0.5, 1, 2, and 3 hours in rat seum, the NGF preparation retained stable bioactivity (++++ or +++), which slightly decreased at 4 hours and 6 hours (++).CONCLUSION: Liposome-encapsulated NGF has stable bioactivity but its preservation at relatively high temperature with high humidity is difficult.Lyophilized liposome-encapsulated NGF exhibits better bioactivity than NGF-liposome suspension after preservation under various conditions.

Aim To investigate the influence of down-regulating adenosine A1 receptor and adenosine A2 A receptor gene expression on proliferation and activation of acetaldehyde-induced hepatic stellate cell-T6 cells through siRNA. Methods Alcoholic liver fibrosis in vitro model was constructed by inducing HSC-T6 cells with acetaldehyde. siRNA targeting A1R and A2AR were designed and synthesized according to its mRNA. The siRNA was transfected into rat HSC-T6 cells by li-posome LipofectamineTM 2000. HSC cell proliferation was measured by MTT. The mRNA levels of A1R, A2AR, α-SMA, Collagen I in the supernatant of the cell culture were measured by Quantitative Real-Time PCR. The protein levels of A1R, A2AR, α-SMA, Collagen I were measured by Western blot. Results A1 R and A2 AR siRNA effectively inhibited the cell proliferation, and they also significantly decreased the levels of A1R, A2AR,α-SMA, Collagen I, suggesting that A1 R and A2 AR might be potential target genes in the alcoholic liver fibrosis. Conclusions Silencing A1 R or A2 AR by RNAi can significantly inhibit the HSC proliferation, A1R and A2AR may be potential therapeutic target genes for alcoholic liver fibrosis.

BACKGROUND: That the nerve growth factor(NGF) is capable of treating peripheral nerve injury has been broadly acknowledged. But it is not sure whether it is able to pass through blood brain barrier(BBB) to act on central nerve system. In this study, the NGF encapsulated in liposome was compared with NGF alone in their abilities of passing through BBB.OBJECTIVE: To compare the dedicated distribution of NGF in different forms using single photon emission computerized tomography(SPECT).DESIGN: It is a randomized controlled study with New Zealand rabbits as subject.SETTING: An affiliated hospital of Nanjing Medical College.MATERIALS:The trial was conducted in Nanjing Senke Medical Company and the Nuclear Medicine Department of the First Hospital of Nanjing Medical College from June 2003 to May 2004. The subjects were 19 New Zealand rabbits of either sex, weighting (2.0 ±0. 2) kg, from Anlimo Technology Company. [99Tcm] -NGF(labeling rate 98.9%, purity 99.7% )was made by Senke Medical Company. Liposome was provided by Shengyang Pharmaceutical College. Urethine solution(200 g/L) was from Pharmacy Department of NanJing Medical Collage.METHODS: [99Tcm]-NGF was encapsulated in liposome and was treated as the following: The liposomesA containing 1.48 × 108Bq[99Tcm] -NGF were injected into rabbits and its distribution percentage was analyzed with SPECT. The same amounts of[99Tcm] -NGF and[99Tcm] -NGF-ordinary liposomesB were treated in the same way.MAIN OUTCOME MEASURES: The ratio of concentration and radiation percentage of NGF in brain to those in the whole body.RESULTS: The[99Tcm]-NGF encapsulated in self-made liposomeA presented high radiation in the brain. But[99Tcm] -NGF alone was almost completely excreted through urinary system and[99Tcm] -NGF encapsuled in ordinary liposomeB was mostly phagocytized by liver reticuloendothelial system.CONCLUSION: The self-made NGF-liposomeA is brain-dedicated, which set a basis for drugs to pass through BBB.

OBJECTIVETo establish a murine model of sensitization, and investigate the effect and mechanism of sensitization on allogeneic donor bone marrow cells (BMCs).

METHODSSensitized BALB/c mice were established by transfusions of allogeneic splenocytes. The donor reactive antibodies were detected by binding and complement-dependent cytotoxicity assays. After irradiation, 1 × 10(7) BMCs of C57BL/6 donor mice were injected into non-sensitized or sensitized BALB/c recipient mice. The distribution pattern of donor BMCs in peripheral blood, spleen and bone marrow of recipient mice were analyzed at different time points (2 h, 12 h and 48 h) post transplantation. Hematopoietic recovery post transplantation was assessed, and survival was monitored. Moreover, sera and splenocytes derived from non-sensitized or sensitized recipients were incubated with allogeneic BMCs in vitro, and the cytotoxic indexes were calculated in the immune experiments.

RESULTSThe binding and complement-dependent cytotoxicity assays showed that a high level of donor reactive antibodies was presented in sensitized sera. Compared with the non-sensitized recipients, the homing assay showed significantly decreased distributions of allogeneic donor BMCs in peripheral blood, spleen and femur of sensitized recipients. Non-sensitized recipients survived long term after irradiation, while all the sensitized recipients died within 12-15 days. Fourteen days post transplantation, the white blood cells and BMCs of non-sensitized recipients were (3240 ± 300) × 10(6)/L and (396 ± 27) × 10(6)/femur, respectively; while the white blood cells and BMCs of sensitized recipients were (320 ± 80) × 10(6)/L and (6 ± 2) × 10(6)/femur, respectively; the differences were statistically significant between this two groups (P < 0.05). Seven days post transplantation, the percentage of donor cells in bone marrow of non-sensitized and sensitized recipients was (48.07 ± 4.70)% and (0.77 ± 0.11)%, respectively, and the differences were statistically significant (P < 0.05). Furthermore, the white blood cells and BMCs following transplantation decreased along with time in sensitized recipients. The immune experiments of complement-dependent cytotoxicity reaction, cytotoxic T lymphocytes reaction and antibody-dependent cellular cytotoxicity showed the cytotoxic indexes were higher in sensitized group than the non-sensitized group.

CONCLUSIONA sensitized model was established by transfusions of allogeneic spleen cells. Allogeneic donor BMCs were rejected in sensitized recipients, and its mechanism might be through immune impairment pathways.

Animals ; Bone Marrow Cells ; Bone Marrow Transplantation ; Disease Models, Animal ; Graft Rejection ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Tissue Donors ; Transplantation, Homologous

Estrogen receptor alpha (ERalpha) has been a primary target of treatment as well as a prognostic indicator for breast cancer. The level of human X-box binding protein 1 (XBP-1) mRNA was related with that of ERalpha in breast tumors and was over-expressed in some breast tumors. These previous studies suggested that XBP-1 may interact with ERalpha. XBP-1 has two isoforms, XBP-1S and XBP-1U, as the result of unique splicing. GST pull-down assay showed that both XBP-1S and XBP-1U bound to ERalpha in vitro. The binding of XBP-1S to ERalpha was stronger than that of XBP-1U to ERalpha. Co-immunoprecipitation revealed that the binding was in a ligand-independent manner. XBP-1S and XBP-1U interacted with the region of ERalpha that contains a DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, the N-terminal basic region leucine zipper domain (bzip) and the C-terminal activation domain. These findings suggest that XBP-1S and XBP-1U may participate in ERalpha signaling pathway through the mediation of ERalpha.
Breast Neoplasms ; genetics ; metabolism ; Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; metabolism ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Humans ; Protein Interaction Domains and Motifs ; physiology ; RNA, Messenger ; biosynthesis ; genetics ; Regulatory Factor X Transcription Factors ; Signal Transduction ; Transcription Factors ; genetics ; metabolism ; X-Box Binding Protein 1

BACKGROUNDIn China the ginseng root began to be used in medicine over 2000 years ago. Ginsenosides are the most important component isolated from ginseng. The authors investigated the effect of ginsenoside Rg1 on the spectrum of gene expression in the endothelial cells stimulated by TNF-alpha and further explored the potential molecular mechanism of endothelial protection by ginsenoside Rg1.

METHODSNitric oxide (NO) production in the cultured human umbilical vein endothelial cells (HUVECs) was measured by using an NO assay kit. A home-made oligonucleotide microarray containing approximately 400 cardiovascular disease-related genes was constructed. The alteration of the spectrum of gene expression induced by ginsenoside Rg1 in HUVECs which were activated by TNF-alpha were detected by oligonucleotide microarray analysis.

RESULTSNO production in HUVECs was decreased significantly after TNF-alpha treatment, while pretreatment with ginsenoside Rg1 enhanced NO production in TNF-alphastimulated HUVECs. Ginsenoside Rg1 affected the expression levels of genes involved in vascular constriction, cell adherence, coagulation, cell growth and signal transduction in TNF-alphastimulated HUVECs.

CONCLUSIONSGinsenoside Rg1 could enhance NO production and the expression of eNOS mRNA in TNF-alpha stimulated HUVECs. Ginsenoside Rg1 regulated sets of genes in endothelial cells and protected endothelial cells from TNF-alpha activation. Microarray analysis provided us with valuable insights into the atheroprotective mechanism by gingsenoside Rg1.

Endothelial Cells ; drug effects ; physiology ; Gene Expression ; drug effects ; Ginsenosides ; pharmacology ; Humans ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase ; analysis ; Nitric Oxide Synthase Type III ; Oligonucleotide Array Sequence Analysis ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo measure the effect of laser fluence and scanning velocity on ablation efficiency of enamel and dentin.

METHODSTwo extracted human incisors and two molars were cut transversely along the axial plane with a diamond saw to obtain dentin and enamel slices with thickness of about 1 mm. Samples were fixed on a motorized translation stage, the linear reciprocating movement in the plane perpendicular to the direction of laser incident was programmed by the controller, and the laser focused on the tooth surface, then 36 ablation lines on enamel and 48 ablation lines on dentin were produced. A femtosecond laser system with wavelength of 800 nm, pulse width 30 fs, repetition frequency 1000 Hz was used, and the diameter of the focused spot was approximately 25 µm. A group of different fluence (1.33, 1.77, 2.21, 4.42, 8.85, 17.69 J/cm(2) for enamel and 0.44, 0.66, 0.88, 1.33, 1.77, 2.21, 4.42, 6.63 J/cm(2) for dentin) and two scanning velocity (10 mm/s and 20 mm/s) were tested. Confocal laser scanning microscope was used to measure the ablation volume.Ablation efficiency for enamel and dentin was then calculated.

RESULTSUnder the fluence of 8.85 J/cm(2) there was the highest ablation efficiency for enamel, 18.703×10(-3) mm(3)/J (20 mm/s), and the highest ablation efficiency for dentin was found under the fluence of 2.21 J/cm(2), ie.223.458×10(-3) mm(3)/J (20 mm/s).

CONCLUSIONSFluence and scanning speed of this femtosecond laser can affect ablation efficiency for both enamel and dentin, and this suggests that with appropriate choice of fluence and scanning speed we can improve the ablation efficiency for enamel and dentin.

Dental Enamel ; radiation effects ; Dentin ; radiation effects ; Humans ; Incisor ; anatomy & histology ; Lasers ; Molar ; anatomy & histology

OBJECTIVETo study gene expression of collagen types IX and X in human lumbar intervertebral discs during aging and degeneration and to explore the role of collagen types IX and X in disc degeneration.

METHODSFetal, adult and pathologic specimens were subjected to in situ hybridization with cDNA probes to investigate mRNA-expressions of types IX and X collagen gene.

RESULTSIn fetal intervertebral discs, positive mRNA hybridization signals of type IX collagen were concentrated in the nucleus pulposus and the inner layer of anulus fibrosus. Interstitial matrix of the nucleus pulposus also showed positive type X collagen staining. Positive mRNA hybridization signals of types IX and X were not detected in the middle and outer layers of anulus fibrosus. In adult specimens, expression of type IX collagen mRNA was markedly decreased. No hybridization signals of type X collagen was observed. As for pathological specimens, there was no gene expression of type IX collagen. In severe degenerated discs from adults, there were focal positive expressions of type X collagen.

CONCLUSIONSObvious changes of collagen gene expression occur with aging. Expression of type IX collagen decreases in adult and pathological discs. Results of type X collagen expression suggest that type X collagen is expressed only in older adult and senile discs (i.e., when disc degeneration has already reached a terminal stage), indicating the terminal stage of degeneration.

Adolescent ; Adult ; Collagen Type IX ; metabolism ; Collagen Type X ; metabolism ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Intervertebral Disc ; embryology ; metabolism ; Lumbar Vertebrae ; Male

Aptamers are capable of binding a wide range of biomolecular targets with high affinity and specificity. It has been widely developed for diagnostic and therapeutic purposes. Because of unique three dimensional structures and cell-membrane penetration, aptamers inhibit virus infection not only through binding specific target, such as the viral envelope, genomic site, enzyme, or other viral components, but also can be connected to each other or with siRNA jointly achieve antiviral activity. Taking human immunodeficiency virus and hepatitis C virus as examples, this paper reviewed the effects and mechanisms of aptamers on disturbing viral infection and replication steps. It may provide an insight to the development of aptamer-based new antiviral drugs.
Antiviral Agents ; pharmacology ; Aptamers, Nucleotide ; pharmacology ; therapeutic use ; Genome, Viral ; drug effects ; HIV ; drug effects ; HIV Reverse Transcriptase ; metabolism ; Hepacivirus ; drug effects ; genetics ; Humans ; Macular Degeneration ; drug therapy ; Neoplasms ; drug therapy ; Oligodeoxyribonucleotides ; therapeutic use ; RNA, Small Interfering ; pharmacology ; SELEX Aptamer Technique ; Viral Envelope Proteins ; metabolism ; Virus Replication ; drug effects

BACKGROUNDThe rocking and instability of a loaded complete denture (CD) during lateral excursion reduce the bearing area under the denture base, causing localized high stress concentrations. This can lead to mucosal tenderness, ulceration, and alveolar bone resorption, and the linear occlusion design was to decrease the lateral force exerted on the denture and to ensure denture stability. But it is not known how the bearing areas of linear occlusal CDs (LOCDs) and anatomic occlusal CDs (AOCDs) differ. The purpose of this study was to analyze and compare the distributions of the high and low vertical stress-bearing areas in the mandibular alveolar mucosa under LOCDs and AOCDs at lateral excursion.

METHODSComputerized tomography (CT) and finite element analysis were used to establish three-dimensional models of an edentulous maxilla and mandible with severe residual ridge resorption. These models were composed of maxillary and mandibular bone structure, mucosa, and the LOCD or AOCD. Lateral excursion movements of the mandible were simulated and the vertical stress-bearing areas in the mucosa under both mandibular CDs were analyzed using ANSYS 7.0.

RESULTSOn the working side, the high stress-bearing (-0.07 to -0.1 MPa) area under the LOCD during lateral excursion was smaller than that under the AOCD, while the medium stress-bearing (-0.03 to -0.07 MPa) area under the LOCD was 1.33-fold that under the AOCD. The medium stress-bearing area on the non-working side under the LOCD was 2.4-fold that under the AOCD. Therefore, the overall medium vertical stress-bearing area under the LOCD was 20% larger than that under the AOCD.

CONCLUSIONSDuring lateral excursion, the medium vertical stress-bearing area under a mandibular LOCD was larger and the high vertical stress-bearing area was smaller than that under an AOCD. Thus, the vertical stress under the LOCD was distributed more evenly and over a wider area than that under the AOCD, thereby improving denture stability.

Aged ; Computer Simulation ; Dental Occlusion ; Dental Stress Analysis ; Denture, Complete ; Female ; Finite Element Analysis ; Humans ; Mandible ; physiology ; Stress, Mechanical