Establishment of Transgenic Mouse Leukemia Cell Lines Expressing Human CD4/CCR5/CyclinT1 Infected with HIV-1.

PURPOSE: Establishing a cross-species animal model of human immunodeficiency virus (HIV) infection is crucial for the study of HIV/acquired immunodeficiency syndrome (AIDS). However, due to the species-specific characteristics of HIV-1, the virus can only infect directly humans and a small number of non-human primates. It cannot directly infect mouse cells across species. METHODS: A mouse leukemia cell line with high CD4 (clusters of differentiation 4)/CCR5 (CC-chemokine receptor 5)/CyclinT1 expression was constructed in this study. First, CD4/CCR5/CyclinT1 lentiviral vector was used to infect a murine leukemia cell line L1210 to express the receptor CD4, co-receptor CCR5 and tat protein driving factor CyclinT1, which are required to infect L1210 cells with HIV-1. RESULTS: The results of sequencing identification and fluorescence expression showed that the plasmid expressing CD4, CCR5, and CyclinT1 vector was successfully constructed and wrapped as the lentiviral vector. Moreover, it was observed that CD4, CCR5, and CyclinT1 proteins were highly expressed in mouse leukemia cells L1210 compared to empty lentiviral vector-transfected cells. Next, viral entry and replication were demonstrated when HIV-1 RNA was detected in body cells and cultured supernatants. Transgenic mice cells L1210 showed significantly greater content of HIV-1 RNA compared to control L1210 cells. Finally, CEMx174 was infected with cell culture supernatants to clarify that the progeny virus is an active virus with infection ability. HIV-1 RNA was highly expressed in CEMx174 cells. CONCLUSIONS: This study made the foundation for future studies evaluating HIV-1 cross-species infection in a murine animal model. The results provided new direction for studies investigating the development of vaccines, antiviral drugs screening, and HIV/AIDS pathogenesis.

Chemo-resistance of A172 glioblastoma cells is controlled by miR-1271-regulated Bcl-2.

MicroRNAs (miRNAs) have been reported to exert important effects on the initiation, progression and metastasis of glioblastoma multiforme (GBM). In this study, we aimed to explore the regulation role of miR-1271 on the development of GBM. We found that miR-1271 was a Bcl-2-targeting miRNA, and the levels of miR-1271was decreased in samples from patients with GBM, compared with those from corresponding normal tissue samples. On the other hand, the levels of miR-1271 were inversely related to the levels of Bcl-2, which have been significantly increased in GBM samples. The overall survival was poorer in patients with low levels of miR-1271, compared to those with high levels of miR-1271. In vitro, the chemo-resistant cell survival mediated with Bcl-2 was inhibited by overexpression of miR-1271 and was enhanced by depletion of miR-1271. Thus, the chemo-resistance of GBM cells may be promoted after suppressing miR-1271 through cell survival mediated with Bcl-2. The prognosis of patients with GBM receiving chemotherapy may be improved by overexpressing miR-1271 in cancerous cells.

Establishment of mouse leukemia cell lines expressing human CD4/CCR5 using lentiviral vectors.

A low-cost rodent model of HIV infection and which presents high application value is an effective tool to investigate HIV infection and pathogenesis. However, development of such a small animal model has been hampered by the unsuitability of rodent cells for HIV-1 replication given that the retrovirus HIV-1 has high selectivity to its host cell. Our study used the mouse leukemia cell lines L615 and L1210 that were induced by murine leukemia virus and transfected with hCD4/CCR5 loaded-lentiviral vector. Lentiviral vectors containing the genes hCD4/CCR5 under the transcriptional control of cytomegalovirus promoter were designed. Transfection efficiencies of human CD4 and CCR5 in L615 and L1210 cells were analyzed by quantitative real-time polymerase chain reaction (RT-PCR) and Western blot assay. Results showed that hCD4 and CCR5 proteins were expressed on the cell surface, demonstrating that the L615 and L1210 cells were humanized and that they possess the characteristics necessary for HIV infection of human host cells. Moreover, the sensitivity of human CD4/CCR5 transgenic mouse cells to HIV infection was confirmed by RT-PCR and ELISA. Mouse leukemia cell lines that could express hCD4 and CCR5 were thus established to facilitate normal entry of HIV-1 so that a human CD4/CCR5 transgenic mice cell model can be used to investigate the transmission and pathogenesis of HIV/AIDS and potential antiviral drugs against this disease.

Pain-Induced Pulsograph Changes in Patients with Primary Dysmenorrhea: A Pilot Study.

Objectives. To investigate changes in pulsograph caused by pain in primary dysmenorrhea (PD) patients. Methods. Pulsograph and pain level of PD patients were detected using electropulsograph and Visual-Analogue Scale (VAS), respectively, at four time points, 7-10 days before menstruation (T0), maximal pain during menstruation (T1), immediately after acupuncture analgesia (T2), and 30 mins after acupuncture analgesia (T3). Parameters (t, h, w) and normalized time parameters (t') of pulsograph were analyzed. Results. VAS pain scores decreased from 6.40 ± 1.13 at T1 to 0.70 ± 0.75 at T2 to 0.11 ± 0.32 at T3 (P < 0.001 and 0.001). At T1, compared with those at T0, w1, h3, and h4 significantly increased (P < 0.01), and t2, t2', t3', and h(d) significantly decreased (P < 0.01, 0.001, 0.05, and 0.001). At T2, compared with those at T1, t1, w1, w2, h2, h3, t1', and t4' significantly decreased (P < 0.05, 0.01, 0.01, 0.001, 0.01, 0.001, and 0.05), and h(d) significantly increased (P < 0.001). There was no difference between T2 and T3. Conclusions. There are almost opposite changing trends in pulsographic parameters when pain occurs and when it is relieved in PD patients.

Confocal mirco-Raman spectroscopic analysis of the antioxidant protection mechanism of the oligosaccharides extracted from Morinda officinalis on human sperm DNA.

ETHNOPHARMACOLOGICAL RELEVENCE: Male infertility is a stressful and frustrating problem for the society, but a number of male infertility treatments are available as traditional Chinese medicine strategies which have been tried with variable success, while evidence is still limited on whether-or how much-herbs or supplements might help increase fertility, so the aim of this study was to investigate if the oligosaccharides extracted from Morinda officialis, a Chinese herb, is the active constituents to the fertility. MATERIALS AND METHODS: In this study, we prepared the H2O2-demaged human sperm, cocultured with the oligosaccharides in vitro, then observed the changes of the DNA using confocal micro-Raman spectroscopy, and comparative analysis the differences of the spectra of different treated groups. RESULTS: The results showed that the oligosaccharides extracted from Morinda officialis can keep the "Raman fingerprints" of the human sperm DNA almost the same as those of the control groups, but very different from the H2O2-induced groups, especially the intensity of bands at 787, 993, 1094, 1254, 1340, 1376, 1421, 1443, 1487, 1577 and 1662cm(-1) which could be as potential targets for the drugs finding, and further principal component analysis was successfully used to classify the Raman spectra of normal control and model groups. CONCLUSION: This results suggested that the oligosaccharides can protect the DNA of human sperm from being damaged by H2O2, and which was one of the active constituents of Morinda officialis on treating infertility. It was also demonstrated that Morinda officialis as a tonifying and replenishing natural herb medicine can be used to enhance reproductive functions, and the Raman spectroscopy could be an applicable technology for screening active components in vitro from herbs.

[Quantization determination study of micro-Raman spectroscopy of methemoglobin induced by sodium nitrite].

In the present study, Raman spectral characteristics of methemoglobin (MetHb) induced by sodium nitrite (NaNO2) were investigated. Hemoglobin (Hb) was oxidated to MetHb with NaNO2, the Raman spectral specific changes of MetHb was studied by determining the Raman spectral changes of methemoglobin/total hemoglobin of different ratios, and the Raman intensities of methemoglobin/total hemoglobin of different ratios at 1 586, 1 605 and 1 637 cm(-1) were linearly fitted to realize its quantitative detection. The results show that the completely oxidized MetHb can be obtained when the molar ratio of NaNO2 to Hb is 3.5 : 1 whose Raman characteristic peaks are at around 499, 1 340, 1 562 and 1 622 cm(-1), and that the linear fitting correlation coefficients R2 of the Raman intensities of methemoglobin/total hemoglobin of different ratios at 1 586, 1 605 and 1 637 cm(-1) are 0.972 84, 0.997 97 and 0.991 26 respectively, which shows a good linear relationship. This study indicates that the Raman spectrums of MetHb induced by NaNO2 have characteristic differences when compared with normal Hb, that the locations and intensities of Raman characteristic peaks change correspondingly with the alterations of the ratios of methemoglobin/total hemoglobin, and that there are linear correlations between the ratios and their corresponding Raman intensities, which would provide theoretical bases for the clinical Raman spectral detection and quantitative study of methemoglobinemia.

[Raman spectroscopy study of the effect of H+ on the oxygen affinity capacity of hemoglobin].

The hemoglobin was extracted from the blood which was provided by the healthy volunteers and the impact of the pH on hemoglobin oxygen binding capacity was studied with microscopic Raman spectroscopy. The results indicated that: under the excitation light of 514.5 nm, with the reducing of the oxygen partial pressure (PO2), the Raman peak intensity at 1 375, 1 562, 1 585 and 1 638 cm(-1) of the control hemoglobin (pH 7.4) reduced gradually, among which, the change of the 1 375 and 1 638 cm(-1) were the most significant and had a good relevance with the PO2. The curves were plotted by regarding the PO2 as the x-axis and the Raman absolute intensity as the y-axis, and the relationship between hemoglobin Raman absolute intensity of the 1 375 and 1 638 cm(-1) and their related PO2 levels when the pH was 5.7, 7.4 and 8.0 respectively were analyzed. The data was well linear fitted and the fitting equation was obtained. The relationship of the slope (Raman intensity/PO2 level) among them were K8.0 > K7.4 > K5.7, indicating that the lower the pH, the easier the release of the oxygen molecules. It was showed that the Raman spectroscopy technique could be used to detect the oxygen binding rate of hemoglobin quantitatively, and the effect of the PH on oxygen binding state of hemoglobin could be observed, which could provide a new method and make a foundation for the monitoring of the PO2 levels in the blood, as well as the research on the regulatory factors of the blood oxygen affinity, such as H+ and CO2.

Comparison of microRNA expression in hippocampus and the marginal division (MrD) of the neostriatum in rats.

BACKGROUND: MicroRNAs (miRNAs), a class of highly conserved small non-coding RNA molecules, are known to play essential roles in central nervous system (CNS) by causing post-transcriptional gene silencing. There is much evidence that miRNAs have specific temporal and spatial expression patterns in the mammal brain, but little is known about the role of the region specificity for the gene regulatory networks of the brain. This study represents the first attempt to perform a profiling analysis of the differential expression of miRNAs between hippocampus and the Marginal division (MrD) of the neostriatum in the rat brain. RESULTS: Microarray was used to detect the expression of 357 miRNAs in hippocampus and the MrD from three rats. A short-list of the most dysregulated 30 miRNAs per rat was generated for data analysis, and the miRNAs that were represented in two or three short-lists were then further analyzed. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed to validate the aberrantly expressed miRNAs obtained from the miRNA microarray analysis. A family of 11 miRNAs demonstrated differential expression between the MrD and hippocampus in more than one rat. Amongst these, miR-383 was differentially expressed in all three rats and up-regulated to the largest degree in rat one, and the ten other miRNAs, let-7d*, miR-181b, miR-187, miR-195, miR-214, miR-382, miR-411, miR-466b, miR-592 and miR-1224 were differentially expressed in at least two rats. Of these ten, besides miR-382 and miR-411 which were up-regulated in one rat and down-regulated in another, the other eight miRNAs retained a uniform direction of regulation (up-regulation or down-regulation) between different specimens. When further examined by RT-PCR, the aberrantly expressed miRNAs, except miR-383 and let-7d*, demonstrated differential expression that significantly correlated with the microarray findings. CONCLUSION: This study reported that the miRNA expression patterns in MrD was distinct from that of Hip, suggesting the role of miRNAs in the learning and memory function of the MrD probably different from hippocampus.

[Chromaticity and optical spectrum colorimetry of the tongue color in different syndromes of primary hepatic carcinoma].

OBJECTIVE: In this study, the optical data of tongue color of different syndromes in primary hepatic carcinoma (PHC) were detected by optical spectrum colorimetry, and the chromaticity of tongue color was compared and analyzed. The tongue color characteristics of different syndromes in PHC and the relationship between different syndromes and tongue color were also investigated. METHODS: Tongue color data from 133 eligible PHC patients were collected by optical spectrum colorimetry and the patients were divided into 4 syndrome groups according to their clinical features. The syndrome groups were liver depression and spleen deficiency (LDSD), accumulation of damp-heat (ADH), deficiency of liver and kidney yin (DLKY), and qi stagnation and blood stasis (QSBS). The variation characteristics of chromaticity coordinates, dominant wavelength, excitation purity and the distribution in the International Commission on Illumination (CIE) LAB uniform color space were measured. At the same time, the differences of overall chromatism, clarity, chroma, saturation and hue were also calculated and analyzed. RESULTS: PHC patients in different syndrome groups exhibited differences in chromaticity coordinates. The dominant wavelength of QSBS was distinctly different from that of the other 3 syndromes. Excitation purity in the syndromes of LDSD, ADH and DLKY showed gradual increases (P<0.01). Different syndromes in the CIE LAB color three-dimensional space showed differences in tongue color distribution areas. The CIE hue-angle value of QSBS was negative, and different from that of the other 3 syndromes (P<0.01). CIE chroma in the syndromes of LDSD, ADH and DLKY showed gradual increases (P<0.01), the same as excitation purity. In the comparison of chromatism, tongue color variations in different syndromes were quantified by human observation. CONCLUSION: This study shows that tongue color diagnosis according to the syndrome classifications of traditional Chinese medicine can be quantified with optical spectrum colorimetry technology. Different syndromes in PHC exhibit distinct chromatisms of tongue color through the calculation and analysis of chromaticity parameters of CIE, combined with colorimetric system and CIE LAB color space, and these are consistent with the characteristics of clinical tongue color. Applying optical spectrum colorimetry technology to tongue color differentiation has the potential to serve as a reference point in standardizing traditional Chinese medicine syndrome classification in PHC.

[Effects of indocyanine green on near-infrared optical properties and optical coherence tomographic image of cerebral blood vessel in vivo in rats].

The scope of this research lies in the effects of indocyanine green (ICG) on the near-infrared optical properties and optical coherence tomographic image of cerebral blood vessel in vivo in rats. The skulls of SD rats were opened under nembutal anesthesia to expose and mark the middle cerebral artery. The reflectance spectra of middle cerebral artery were monitored by Vis/ NIR spectrometer and the optical attenuation coefficients of middle cerebral artery were detected by optical coherence tomography (OCT) when indocyanine green was administrated intravenously through tail veins. It was shown that the reflectance spectra of middle cerebral artery could provide guidance for OCT image, where characteristic changes appeared around 800 nm, an absorption peak of indocyanine green. Additionally, significant difference (p<0.01) was observed between the optical attenuation coefficients of middle cerebral artery with and without indocyanine green, which were 24.692 +/- 1.471 and 15.088 +/- 1.602, respectively. It was concluded that indocyanine green, as an optical contrast agent to enhance detection of cerebral artery by the reflectance spectra and OCT imaging, has the potential for monitoring and imaging of cerebral blood vessels.

[Influence of brightness value of supranasal point and apex nasi on dominant wavelength and excitation purity in complexion inspection of healthy adults].

OBJECTIVE: In this study, to analyze the influence of the brightness value of the supranasal point and the apex nasi on their dominant wavelength and excitation purity according to the spectrocolorimetry data of the supranasal point and the apex nasi in healthy adults that were collected based on optical spectrum colorimetry. METHODS: A total of 516 healthy adults were taken as the research subjects. The brightness, dominant wavelength and excitation purity values of the supranasal point and the apex nasi during the complexion inspection of subjects were calculated. This was based on the visible reflection spectrum, and the linear correlation/regression analysis between the brightness Y value and the dominant wavelength or excitation purity value. RESULTS: There was no correlation between the brightness Y value and the dominant wavelength of the normal supranasal point and the apex nasi; however, there was negative correlation between the brightness Y value and the excitation purity of the normal supranasal point and apex nasi. CONCLUSION: During the complexion inspection, the brightness Y value would not influence the dominant wavelength value, indicating that whiteness and/or blackness would not influence the normal individual complexion. However, the brightness Y value would influence the excitation purity of the supranasal point and the apex nasi, and the degree of saturation should be referred to as the brightness. This research provides a basic reference for diagnosing facial complexion in traditional Chinese medicine.

[Colorimetric investigation of normal tongue and lip colors from 516 healthy adults by visible reflection spectrum].

OBJECTIVE: Using the data from normal tongue and lip colors of normal people which were collected by the visible reflection spectrum, we analyzed the colorimetric parameters of tongue and lip colors. METHODS: In this study, 516 healthy students aging from 19 to 26 from the colleges and universities of Guangdong Province of China were taken as research subjects. After collecting the data of tongue and lip colors of the 516 subjects using visible reflectance spectroscopy, CIE XYZ tristimulus values as defined by the International Commission on Illumination in 1964 were calculated, and the colorimetric parameters of the normal tongue and lip colors were obtained, such as the CIE 1964 chromaticity coordinate, brightness, dominant wavelength and excitation purity. RESULTS: The results of CIE 1964 chromaticity diagram calculated on the visible reflection spectrum showed that the normal tongue color chromaticity coordinate x(10) was 0.341 3±0.008 5 and y(10) was 0.332 6±0.005 1, and the normal lip color chromaticity coordinate x(10) was 0.357 7±0.009 2 and y(10) was 0.338 3±0.005 7; the brightness Y values of the normal tongue color and lip colors were 17.96±3.78 and 19.78±3.72, the dominant wavelength values of the normal tongue color and lip color were (626.3±51.6) nm and (600.4±18.2) nm, and the excitation purity values of the normal tongue color and lip color were 0.083±0.031 and 0.144±0.036, respectively. CONCLUSION: Application of the visible reflection spectrum is a standard way to collect colorimetric data for inspection of the complexion. The investigation of chromaticity coordinates, brightness, dominant wavelength and excitation purity of the normal tongue and lip colors may offer the basic reference for diagnosing morbid complexion on the tongue and lip colors in traditional Chinese medicine.

The role of substance P in the marginal division of the neostriatum in learning and memory is mediated through the neurokinin 1 receptor in rats.

Substance P (SP) is a neuropeptide that plays an important role in inflammation, respiration, pain, aggression, anxiety, and learning and memory mainly through its high affinity neurokinin 1 receptor (NK1R). The marginal division (MrD) is a pan-shaped subdivision in the caudomedial margin of the neostriatum in the mammalian brain and is known to be involved in learning and memory. We studied the expression of SP, NK1R and NK1R mRNA in the rat striatum by immunohistochemistry, immunofluorescence and in situ hybridization, and found that the levels of SP, NK1R protein and NK1R mRNA were high in the cell bodies, fibers and terminals of neurons in the neostriatum, especially in the MrD. Knocking down NK1R activity in the MrD by using an antisense oligonucleotide against NK1R mRNA inhibited learning and memory in a Y-maze behavioral test. Our results show that NK1R mediates the role of SP in the MrD in learning and memory.

[Quantitative analysis and classification of tongue inspection based on the visible reflection spectrum].

OBJECTIVE: In this study, optical data on color of tongue tips were collected by using a visible reflection spectrum system. Quantitative method of tongue inspection and classification of tongue states including pale, pale red, red and crimson manifestations were investigated. METHODS: Chromaticity coordinates of the tongue tips from 98 subjects were calculated on the basis of the visible reflection spectrum. The tongue color was quantified and classified by the visible reflection spectrum in the range from 590 nm to 780 nm, based on the higher red-band energy distribution on the tongue tip color. RESULTS: It was found that comparison and analysis of tongue tip color from different samples would be well-suited after spectrum vector normalization, and the difference of tongue tip color between different samples could be observed easily when the tongue tip optical data were sited in CIE 1931 chromaticity diagram. The tongue tip colors were analyzed quantitatively and classified by the spectral energy intensity at the wavelength of 670 nm and (or) spectral energy ratio of red bank from 590 nm to 780 nm. The results of classification showed that there was sound corresponding accuracy with the clinical diagnosis of pale tongue, pale red tongue, red tongue and crimson tongue. CONCLUSION: In this study, based on the collection of the information data on tongue tip characteristics with the visible spectrum from 380 nm to 780 nm, the quantitative analysis of tongue inspection for pale tongue, pale red tongue, red tongue and crimson tongue was established. The quantitative value for tongue tip color is an effective method for classification of the condition of the tongue tip, which provides a useful tool for the modernization of tongue inspection in traditional Chinese medicine.

[Comparative study of reflectance spectroscopy of women's acupoints around menstruation].

Acupoint was reaction of viscera and its optical parameter was an important characteristic of tissue. In the present work, in order to discuss specificity of acupuncture on the response of qi and blood in human body, we compared the diffuse reflectance of Taichong (LV3), Taibai (SP3), and Chongyang (ST42) before, during and after menstruation. All the acupoints had the same shape of the spectrum and troughs were all at 423.16, 544.06 and 577.47 nm. The values of reflectance during menstruation were greater than before and after menstruation, especially for SP3 and ST42 (P < 0.05). SP3 and ST42 were more sensitive to the changes of qi and blood than LV3, and it was asymmetric and we found no evidence for the imbalance of the left side and right. These results indicated that acupoint diffuse reflectance changed with qi and blood, and that SP3 and ST42 had close relationship with menstruation. Diffuse reflectance may be possibly used in the quantitative analysis of qi and blood.